Chapter 10: Principles of Development in Vertebrates PDF

CHAPTER 10 In the last two blocks of this course you learnt about the diversity in the anatomy of vertebrates. In the following units of this course we will learn about the progressive period of animal development during which the various organ systems develop in vertebrates. In order to achieve th...

CHAPTER 10 In the last two blocks of this course you learnt about the diversity in the anatomy of vertebrates. In the following units of this course we will learn about the progressive period of animal development during which the various organ systems develop in vertebrates. In order to achieve this you will study how various processes occur in the development of mature male and female gametes, called sperms and ova (or eggs) respectively in vertebrates. You will also become familiar with the events that occur prior to and during the period of fertilization (union of sperms and ova) which results in the formation of a single celled zygote that consists of genetic material from both parents. This zygote through various stages and cell processes develops into a multicellular organism that is capable of functioning, growing, reproducing and completing its life cycle. The process of development of the zygote from a single celled entity to a multicellular embryo is long. In humans as you may be aware the duration of development of the zygote into a fully developed foetus, ready to be born is approximately nine months. Block 3 Developmental Biology of Vertebrates-I 10 In the present unit you will study about the principles of development which are common in all organisms both nonchordates and chordates. You will also come to know about the emergence of the field of embryology and how it progressed into the modern and vast discipline of Animal Developmental Biology, due to newer and better biological techniques and the advent of newer biological sciences like molecular biology, genetics etc. This unit will also focus on the various nonchordate and chordate animal models that have been used in the past and are being used in the present study of embryology and animal developmental biology. These animal models have been used in order to understand how animals develop both at the observable, morphological, experimental level and at the level of underlying molecular biology and genetics. You will be able to understand the reasons for choosing a particular animal model in order to study a particular process of development Developmental biology addresses some of the big questions that arise from the study of embryological processes such as: how does a single cell-the fertilized egg- give rise to a multicellular organism in which there are a multitude of different cells that give it form? How do the various cell types -- muscle cells, blood cells, skin cells, neurons etc., - form and get differentiated from one another? How do these cells then get organized into functional organs in the animal body and what can influence these pathways of development? Along with this you will learn about some crucial experiments that gave the developmental biologists insight into these processes. ObjectivesObjectives Objectives Objectives After studying this unit you should be able to: differentiate between Embryology and Developmental Biology, stating the progressive stages in development of a multicellular organism; describe the features of the various nonchordate and chordate animals used as model organisms; explain the role of fate maps and patterns of development; state the concepts of cell specification, determination and differentiation; explain the concept of genomic equivalence, totipotency and pluripotency through classic experiments of Briggs and King; discuss the mechanism of differential gene expression. 10.2 DEVELOPMENT STAGES COMMON TO ALL ANIMALS The blueprint of development of an organism from a fertilized egg to an adult is encoded in (i) genes present in the zygote and (ii) some special clues in the form of cytoplasmic determinants present in the cytoplasm of the zygote. During the course of development, the developing cells of the zygote Unit 10 Principles of Development 11 differentiate into many cell types that communicate and coordinate the various developmental activities and then subsequently get organized to form an integrated functional organism. A development principle common to all higher organisms is that, the fertilized egg or zygote will develop progressively during several stages that last for different periods in order to form an integrated functional organism. The stages of development that occur between fertilization and the birth of an organism (Fig.10.1) are collectively known as embryogenesis. It is during embryogenesis that the genotype (genes of the developing organism) of the organism determines the morphological appearance (phenotype) of the organism. Each animal whether it is a fruit fly or earthworm or frog, or bird or a mammal undergoes the same basic stages of development which include: i) Fertilisation -- involves the fusion of mature male and female sex cells or gametes. Each gamete has only half the complements (set) of the chromosomes of the adult organism and union of the male and the female gamete to form the zygote restores the full genetic complement. The full genetic complement of the zygote instructs the zygote to develop in a similar manner to the parents and to produce an organism similar to the parents. ii) Cleavage or rapid cell division -- is the stage in which the zygote is divided into numerous small cells known as blastomeres. During cleavage cells do not grow between each division and so with successive cleavage cells the blastomeres become smaller. These smaller blastomeres develop into early stages of development called morula and blastula stages. iii) Gastrulation -- is the stage during which the cell division slows down and cells of the blastula undergo dramatic movement and rearrangement causing cellular diversity and formation of the three germ layers namely, ectoderm, mesoderm and endoderm. These three layer interact to form the organs of the body. iv) Morphogenesis -- is the process of cellular differentiation in the embryo. Morphogenesis gives the embryo its shape. v) Organogenesis -- is the process in which organ formation is completed so that the embryo becomes functional. A fully developed individual organism can then be distinctly seen as a member of a particular species at this stage. The embryo then, takes birth as a formed individual that lives on, till its death. In many species a group of cells are set aside and do not participate in the formation of the embryo. These are known as germ cells and are used to produce the next generation. All other cells of the body are known as somatic cells. The germ cells migrate in the embryo to form the gonads and give rise to gametes in the adult organism. However, the process of development does not stop at birth, it is seen as metamorphosis and regeneration in some animal groups and finally as aging or senescence. Block 3 Developmental Biology of Vertebrates-I 12 In Figure 10.1 we have shown the life cycle of frog as an example depicting all the above mentioned stages of development. Fig 10.1: Life cycle of a frog, showing, the various stages from fertilisation and embryogenesis. In the frog the egg hatches as a larva (tadpole) and completes the rest of development through steps of metamorphosis; finally emerging as the adult frog capable of starting another generation. 10.3 HISTORICAL BACKGROUND OF DEVELOPMENTAL BIOLOGY History of animal development is believed to have begun in the 4th century BC with simple observations of egg and embryos that could be seen with the naked eye. Aristotle was the first to record variations in life cycles of animals. He noted that some animals are born from eggs (oviparity) as seen in most invertebrates and some vertebrate groups like frogs and birds. He also noted that in some animals, embryos were born directly as young ones (viviparity) as seen in all mammals except for monotremes. He furthermore, observed that some animals were born from eggs that hatched within the body (ovoviviparity) as seen in some snakes and sharks. Aristotle also observed two types of division patterns in the fertilized egg as the embryos undergo cleavage namely: 1) holoblastic division where the entire embryo divides to form smaller cells as seen in frog and mammals, and 2) meroblastic division pattern of division where only a part of the fertilized egg divides and the rest provides nutrition for the embryo as seen in the chick. Aristotle, on the basis of his studies on the development of chick embryo, for the first time advanced the theory of epigenesis (meaning: 'determination/ upon formation') for the development of organisms. According to the epigenesis theory, new structures develop progressively in the embryo during development. Thus, according to this theory there was no preformed tissue or organ in the embryo at the beginning of development. Unit 10 Principles of Development 13 However, the theory of epigenesis was not the only theory of animal development. The more popular theory that emerged later was that of preformation of embryo in development. According to the preformation theory, all the parts in the embryo were preformed and they just got bigger with time. Preformationists believed that a preformed miniature, that is a fully formed infant, called the homunculus (Fig.10.2), existed within the germ cell of one of its parents, prior to fertilization and this would grow and enlarge into its full form during gestation until ready to be born. The Preformationists were thus either spermists or ovists. Spermists believed that the homunculus existed in the sperms while the ovists believed they were contained in the eggs. Very little progress was made in the field of embryology and it was only in 1651 that William Harvey concluded that all animals arise out of eggs. He was the first to see the blastoderm of the chick which is the clear, yolk free germinal disc, also called the blastodisc and is in the form of a single layer of embryonic epithelial tissue and gives rise to the chick embryo. William Harvey saw the red dots of pooled blood that arose before the formation of the heart or vessels. The two theories preformation and epigenesis were debated till the 17th and early 18th century. Kasper Freidrich Wolff, extended Aristotle's observations and supported epigenesis which disputed the idea of preformation. He observed the developing embryo of chick and demonstrated that the embryo takes its form from tissues that are not seen in the adult organism. The heart, intestine and blood cells all could be seen forming as new in each embryo and there were no preformed miniature organs. The final end to the preformation theory came only in the 1820s when the cell theory came in existence. The cell theory in combination with newer staining techniques and improved microscopy contributed to the development of the discipline of descriptive embryology. Christian Pander (1794-1865) first recognized the existence of three germ layers (ectoderm, mesoderm, and endoderm) in the chick embryo. He also wrote about the interdependence of these layers in forming the embryo. A few years later Martin Rathke (1793-1860), another embryologist discovered layers of cells similar to what Pander had described in the crayfish and put forth the idea that three germ layers were not only found in vertebrates but also in invertebrates. While studying embryos of vertebrates, embryologists discovered that there were many embryonic similarities between vertebrates belonging to different groups. Karl Ernst Von Baer, in 1828 proposed this to be an evidence of evolution and put forth his four laws of animal development; Von Baer described his laws of embryology in his book Über Entwickelungsgeschichte der Thiere \[On the Development of Animals, published in 1828 and 1837\]. In his work, von Baer reviewed existing information on the development of vertebrates. He used the information in this review to extrapolate his laws. These laws, translated by Thomas Henry Huxley in Scientific Memoirs are as follows: Fig.10.2: Pre formation theory depicted by homunculus. Block 3 Developmental Biology of Vertebrates-I 14 1) The more general characters of a large group appear earlier in the embryo than the more special characters 2) From the most general forms the less general are developed, and so on, until finally the most special arise 3) Every embryo of an animal form, instead of passing through the other form, rather becomes separated from them 4) Fundamentally therefore the embryo of the higher form never resembles any other form but only its embryo The explanations of the four laws given by Von Baer are as follows: i) The first law meant that in embryos of an animal group, the general characters develop first before the specialized characters develop. His first law thus contradicted the preformationist theories. ii) The explanation of his second law is that, embryos develop from a uniform and noncomplex structure into an increasingly complicated and diverse organism. For example, a defining and general feature of vertebrates is the vertebral column. This structure thus, appears early in the embryonic development of vertebrates. Other features those which are more specific to a group within the vertebrates, such as hair on mammals or scales on reptiles however, form later during development. Von Baer thus, concluded from the first two laws that development occurs through epigenesis, and the complex form of an animal arises gradually from unformed material during development and not from preformed structures. iii) Von Baer\'s third law referred to the fact that animals from different species start to develop in a similar manner but become more dissimilar from one another as ontogeny (the origination and development of an organism, usually from the time of fertilization of the egg to the organism\'s mature form---although the term can be used to refer to the study of the entirety of an organism\'s lifespan) proceeds. As an example, von Baer discussed the embryos of humans, fish, and chicks, all of which appear similar to each other in the early stages of their development. As they grow, however, they look increasingly different from one another. The embryo of one species never resembles the adult of another species. Von Baer\'s third law thus, theorized that animal embryos diverge from one or a few shared embryonic forms. iv) The fourth law meant that the stages of development in more complex animals never represent the adult stages of less complex animals. In the second half of the 19th century another professor in Germany Ernst Haeckel extended the theory of recapitulation which was stated as the 'biogenetic law'. He proposed that "Ontogeny repeats Phylogeny". This means that the stages of ontogeny (development) of the organism replay that organism's evolutionary history. The biogenetic law has been widely disputed, though Von Baer's laws are generally believed to have been responsible for the progress of developmental biology in the twentieth century. Unit 10 Principles of Development 15 The tools used by early embryologists were simple. Embryological studies involved the use of moulds made of wax or glass strips, direct observation of embryos of various animals, and their manipulation with the help of glass needles which were also used to move and pick embryos. Glass pipettes were also used to pull the embryo by narrow tubes for transfer. Embryologists of that time faced a difficult task in manipulating the embryos and ensuring their survival without them being infected, before they could be isolated, removed and transplanted. Later on with the development of better tools and microscopes with better resolution the "Era of Experimental Embryology began". By the1880s, some embryologists began focusing on various experimental methods in order to understand embryogenesis. For this purpose the embryologists used physical manipulation of the embryo rather than just observing and describing them. The German evolutionary biologist, August Friedrich Leopold Weismann (1834-1914) put forward a model of development in which he assumed that the nucleus and cytoplasm of the zygote contained some special factors or determinants. He proposed that during cleavage these determinants would be unevenly distributed in the dividing cells and so could control future development. The fate of each cell according to him was, therefore, predetermined in the egg by these factors. He called this type of development mosaic. His theory was supported by the experiments conducted by the German Zoologist, Wilhelm Roux (1850-1924), in late1800s on frogs. Wilhelm Roux in his experiments destroyed one of the blastomeres of the frog embryo after the first cleavage by using a hot needle. He found that the other blastomere formed half of the embryo (Fig.10.3). He thus, concluded that development was based on mosaic mechanism and the cells have their characteristic fate determined at each cleavage. Fig.10.3: Experiment done by Roux that supported Weismann's theory of mosaic development. Another German biologist Hans Adolf Eduard Driesch (1867-1941) found something quite opposite; he used sea urchin eggs and separated them after the first cleavage. He found that one blastomere died and the other formed a smaller but complete larva (Fig.10.4). He called this type of development regulative, referring to the ability of the embryo to restore normal development even though one cell had died. We will learn more about determinants and these two types of development namely mosaic and regulative when we discuss specification in cells. Block 3 Developmental Biology of Vertebrates-I 16 Fig.10.4: Driesch's Experiment with sea urchin blastula that demonstrated regulative development for the first time. a) normal development of sea urchin larva from two cell stage; b) separation of cells at two cell stage resulted in the death of one cell but the other survived to develop into a fully formed smaller larva. The next major step in experimental development biology came in 1924 when German embryologist, Hans Spemann (1869-1941), and his graduate student Hilde Mangold proposed the concept of 'organizer' and the principle of embryonic induction during development as seen in the development of amphibian embryos. Their work (Fig.10.5) showed that, in the earliest stages, the fate of the embryonic parts is not determined and if a piece of presumptive skin tissue is excised and transplanted into an area of presumptive nervous tissue, it will form nervous tissue, not skin. It provided the first unambiguous evidence that cell and tissue fates can be determined by signals received from other cells. This experiment is probably the best known in embryology. The groundbreaking and technically demanding experiment was performed in newt embryos at the gastrulation stage, the period during which the three primary germ layers the ectoderm, mesoderm and endoderm become established. The experiment involved transplantation of a structure present on the dorsal side of the blastopore stage of the embryo, called the dorsal lip, to the ventral side of another embryo. By grafting tissue between differently pigmented Triton species, the fates of the graft and host tissues could be distinguished. This graft, nowadays referred to as the Spemann organizer (also the Spemann--Mangold organizer) had two effects. It induced the formation of neural tissues (neuralization), from the ectoderm that would have generally formed the skin. Furthermore it caused dorsalization of the ventral mesoderm, leading to the formation of somites. This experiment therefore, demonstrated the existence of an organizer that instructs both neuralization and dorsalization, and showed that cells can adopt their developmental fate according to their position when instructed by other cells. The molecular nature of this signal remained elusive until 65 years later and is still not completely understood. Hans Spemann was awarded the Nobel Unit 10 Principles of Development 17 Prize, in 1935, for his discovery of the effect now known as embryonic induction Fig.10.5: Experiment conducted by Spemann and Mangold that demonstrated induction of a new main body axis by the organizer region in early amphibian gastrula. a) dorsal lip of blastopore from unpigmented species grafted on the blastocoels roof of a pigmented species; b) a secondary embryo is induced. Towards the middle of the 20th century, the scientific discipline of embryology began to emerge as the modern discipline of developmental biology. As is true for other disciplines in biology, knowledge of animal development advanced: (i) as new techniques for experimentation were invented and (ii) with the progress in knowledge of other branches of biology such as cell biology genetics, and especially molecular biology with the discovery of DNA, and the processes of transcription, translation and gene regulation. With the increase in the knowledge of molecular biology, development biology emerged as a field of study which attempted to correlate genes with the morphological changes that were observed in embryos undergoing development. Which genes were causing which morphological changes and how these genes were controlled to express at certain stages of development became an exciting subject for research for many biologists. It thus became clear that genes which all embryonic cells contained, switched on and switched off as and when required in the developing embryo. More recently, experiments on gene expression have demonstrated clearly that even in widely different animals, body plans share many basic features and mechanism of development. They are controlled by a common set of regulatory genes which direct cells to become capable of distinct functions. For example, the position for formation of eyes in a vertebrate like the mouse has a close counterpart with a nearly identical function in the fruit fly Drosophila which is an invertebrate. Research in animal embryology and subsequently in animal developmental biology advanced with the use of a number of nonchordate and chordate Block 3 Developmental Biology of Vertebrates-I 18 model organisms for studying the development in laboratories. These models were chosen because:1) They had short life cycles,2) were easily available and 3) and were easy to manipulate. Studies using different animal models clearly demonstrated that there are many similarities between the invertebrates and vertebrates during their development. Animal Developmental Biology has now jumped far ahead of where it began in the early 20th century. The tools have changed, certain concepts continue to hold good and new concepts have gained ground. Embryology has travelled far ahead to get the new name Developmental Biology. If a difference between the two was to be sought then it can be conveniently said that "all embryology is developmental biology but some developmental biology is not embryology" SAQ 1 a) Give the difference between embryology and developmental Biology. b) What were the two theories of development? Which theory describes our current view of development? 10.4 ANIMAL MODELS IN DEVELOPMENTAL STUDIES As experimental embryology advanced in the 20th century, scientists observed that there are remarkable similarities in the developmental mechanisms of all animals. Not only genes and proteins but entire signaling pathways and their response in developing embryos appear highly conserved throughout evolution. More recently, experiments on gene expression during embryogenesis have demonstrated that even in widely different body plans of various kinds of animals many basic mechanisms of development are controlled by a common set of regulatory genes. They direct the cells to become capable for performing distinct functions in the developing embryos. For example, gene specifying position for formation of eyes in the mouse (vertebrate) has a close counterpart with a nearly identical function in the fruit fly Drosophila (invertebrate). Scientists, however, found that only a limited number of animal species could be utilized for investigating the developmental processes. These animal species were chosen as model organisms for experimentation earlier in the field of embryology and animal developmental biology. We can define animal model organisms as non- human species that have been widely studied, mostly because: i) of the ease with which the different experimental techniques can be conducted, ii) of the ease with which they can be maintained, iii) of the ease of breeding them in the laboratory iv) of having a short life cycle, Unit 10 Principles of Development 19 v) of having rapid development, vi) of the fact that their robust embryos are easy to manipulate vii) these models may be representative, for answering common and specific questions of development across animal groups. For example, they have been used to obtain answers about whether the mechanism of eye formation in Drosophila maybe considered representative of most vertebrates. Though, the ability to manipulate the model organism is a critical criterion for its selection, it also has to be combined with other considerations such as how easy it is to initiate an experimental set up with an animal model, in terms of cost and availability of the animal models and of availability and suitability of the infrastructure. For example, chick embryo is an established model organism that is easy to manipulate but the inability to apply genetical methods to the chick embryo has limited its use in recent times. The development of newer physical tools and techniques has ensured that organisms like sea urchin and amphibians (in the form of different species of frogs and salamanders) and still later the fruit fly, Drosophila and relatively recently the round worm Caenorhabdites elegans and the zebra fish Danio have become popular experimental models for the study of the various processes and mechanisms of development. Recent developmental studies have moved from the overall, gross study of the embryonic development to studies of the cellular and molecular aspects of animal development. In other words animal development studies have moved from macro level to micro level. Let us examine the characteristics of some of the model organisms that are particularly useful for experimentation in developmental biology. 10.4.1 Classical Models: Sea Urchin and Frog The earliest animals used for study of development were sea urchins, and frogs because of their large eggs which could be handled and observed under the microscope. Their suitability depended on i) easy accessibility; ii) ease of manipulation and iii) easy visualization with the available tools and techniques Sea urchin Historically sea urchins have been a key model system for finding out answers in developmental biology. Sea urchin is an echinoderm and is found in the shallow tide pools in oceans of the world. The features that make it suitable for study are: 1. Gametes can be obtained easily and artificial spawning and fertilization and rearing is possible. 2. Lots of large eggs are produced which are easily visible (Fig.10.6). 3. Early development is highly synchronous. Large numbers of synchronously developing embryos can be obtained quickly and easily. 4. Low maintenance costs due to high fecundity. The gravid adults produce enormous numbers of gametes that are easy to collect in the laboratory. Fig. 10.6: Sea urchin egg seen under the microscope with sperms surrounding it, for fertilization. Block 3 Developmental Biology of Vertebrates-I 20 5. A single female can provide many millions of oocytes and a single male many billions of sperm. Both types of gametes are fully competent to carry out fertilization without additional maturation. 6. Fertilization and embryonic development occur externally, both in nature and in the laboratory. Development is initiated synchronously by simply mixing sperm and eggs. 7. Embryonic development is rapid. In many warm-water species, only 1--2 days are required for the fertilized egg to develop into the pluteus larva. One practical consequence of this rapid development is that the time between experimental manipulations and analysis of their developmental effects is very short. 8. The embryo has a simple structure. At the late blastula stage, the embryo is a simple, hollow ball composed of about 800 cells surrounding a central cavity (the blastocoel). The number of cells approximately double by the end of gastrulation but do not exceed 2000--3000 cells during pre-feeding development. The embryo and pluteus larva have relatively simple morphologies and only 15--20 distinct cell types arise during embryogenesis. 9. The embryo is highly transparent. The embryos of many species of sea urchins are almost as clear as glass. This transparency, coupled with the anatomical simplicity of the embryo, helps in light microscopic observations of development. Thus biologists have constructed a very detailed picture of sea urchin embryogenesis at the cellular level. 10. After microscopic studies to see fertilization and early stages of embryo, the bio chemical and molecular events have also been studied. Small molecules can be used to perturb development. 11. Diverse tools are available for analyzing and manipulating gene expression. Sea urchins are well suited to all modern approaches for analyzing gene expression Recent sequencing of its genome has made it a model organism for study of developmental genetics. Though it is an echinoderm (invertebrate) it encodes many vertebrate immune system related genes. 12. Maternal molecules are specially restricted and are involved in determining cell fate, apoptosis, autophagy and recently are seen to have a role in cellular and molecular mechanisms involved in human health and disease. 13. Over a century, sea urchin has been a prototype for investigation of development of body plan, cell adhesion, cell signaling and age related pathways and mechanisms of cell death. Frog As animal model organisms amphibians offer several advantages because they have a well understood morphology and physiology. The taxonomical position of amphibians is well suited for the comparative developmental studies of reptiles and birds as well as mammals. Developmental studies of Unit 10 Principles of Development 21 frogs and salamanders have made possible important insights into molecular mechanisms that control embryonic cell division, differentiation and animal development. Salamanders serve as important vertebrate models for the study of regeneration and tissue repair. However, the most important models for studies of early vertebrate development have been the African clawed frog Xenopus levis and Rana pipens due to the following practical advantages: 1) Eggs of Xenopus and Rana are large sized and the eggs develop outside the body of the mother and are laid in water. This makes it easy for researchers to collect them from water and carry them to the laboratory so that microscopic studies become possible. 2) They can be easily bred in captivity and be induced to spawn artificially by chorionic gonadotropin. Furthermore, the large number of eggs produced at a time, facilitate biochemical analysis. 3) Their embryos can be observed easily. Xenopus has a short time of development (4 days) before it reaches the free swimming larval stages. 10.4.2 A Later Model: Drosophila Since the middle of the 20th century, the fruit fly Drosophila melanogaster (Fig.10.7) has been a favourite model for research in genetics. After the extensive use of Drosophila for genetic analysis, developmental biologists began to use it as an experimental model for development which led to advances in understanding the process and mechanism of animal development. Drosophila has today at the level of genes become a crucial model organism in developmental biology especially in order to understand gene activation, gene regulation, differential gene expression, genomic equivalence, genetic control in the process of animal development. The advantages of Drosophila as a key model for laboratory studies for genetic and developmental biology are as follows: 1) Drosophila can be easily cultured, maintained and bred in the laboratory. 2) It has a short reproductive cycle (Fig.10.7) of approximately of two weeks and produces many offsprings. The embryo develops outside the mother's body and so can be easily observed under the microscope 3) Drosophila genome contains only about 15,500 genes. 4) Genetic analysis of the fruit fly has unraveled many genes that control development and differentiation. There is a close relationship between Drosophila and vertebrate genes especially human genes that operate during development. For example, it has been found that the homeotic genes that are master regulators as they direct the development of particular body segments are highly conserved in animals and have corresponding genes in fruit flies, mice and humans. 5) Drosophila as a model organism has been very important in research work for information on genetic and molecular basis of animal development as mutants are readily available. Mutants can also be Block 3 Developmental Biology of Vertebrates-I 22 induced to be formed. The mutants have been helpful in studying the powerful role of the genes in directing normal and abnormal animal development. 6) Even more detailed genetic analysis of the development processes has been possible due to current techniques of molecular biology. Fig.10.7: Drosophila life cycle. 10.4.3 Newer Models: C elegans and Danio rerio Two other organisms now being used by developmental biologists are the round worm Caenorhabditis elegans and the zebra fish Danio rerio. Caenorhabditis elegans: 1) Caenorhabditis elegans is a free living nematode that lives in soil but is easily grown in perti-dishes (10.8a). 2) It is only a millimeter long transparent worm with limited cell types (only 959 somatic cells in the body). 3) The adult and embryo are transparent. It requires only 3.5 days to grow from zygote to mature adult and has a large brood size. 4) Embryonic development takes place within 12 hours. 5) Its genomes have been completely sequenced. 6) C elegans was the first metazoan genome to be sequenced. 7) C elegans has a simple system, and so by following the division of its cells, scientists have worked out the complete cell lineage which is invariant. Invariant means that all individuals of the species have the same anatomy. Zebrafish (Danio rerio) 1) The zebrafish is small and robust and cheaper to maintain than mice. 2) Danio rerio, has been chosen for its unique combination of large, easily Unit 10 Principles of Development 23 accessible eggs and embryos that are transparent which allows researchers to easily examine the development of internal structures. Every blood vessel in a living zebrafish embryo can be seen using just a low-power microscope. Thus, changes can actually be observed in the living organism (Fig.10.8 b). 3) Zebrafish produce hundreds of offspring at weekly intervals, providing scientists with an ample supply of embryos to study. The eggs are fertilised and develop outside the mother's body and also serve as an ideal model organism for studying early development 4) Embryos of zebra fish grow at an extremely fast rate, developing as much in a day as a human embryo develops in one month. Although generation time in zebra fish is 2-4 months, the early stages of development take place quickly. By 24 hours after fertilization most tissues and early versions of the organs are formed. After 2 days the fish hatches out of the egg. 5) Zebrafish share 70 per cent of genes with humans. The zebrafish genome has been fully sequenced and the model is also useful for molecular studies Fig.10.8: a) The free living nematode C. elegans; b) early embryonic development of zebrafish eggs and embryos. SAQ 2 a) List three classical animal model used by investigators to study development in animals. What are the common features in them that make them good animal models for studying animal development. b) Which model organism has provided information of genetic control of development through the study of its mutants? (a) (b) Block 3 Developmental Biology of Vertebrates-I 24 10.5 EMERGENCE OF PATTERNS In the beginning of this unit we had written that one of the most fascinating questions in animal development is the generation of a complex organism from a single fertilised cell the zygole. As embryonic development proceeds it involves the emergence of a pattern which shows the overall position of cells in the embryo so that the body's shape and form begins to emerge. This is followed by cell differentiation and growth. Development is a gradual process by which a complex multicellular organism arises from a single cell (the zygote). It involves 5 major overlapping processes: 1) growth = increase in size 2) cell division= increase in number 3) differentiation = diversification of cell types 4) pattern formation = organization 5) morphogenesis = generation of shapes and structures 10.5.1 Pattern Formation As the zygote divides it undergoes a process by which the initially equivalent (similar) cells acquire different identities which depend on their relative positions in the embryo at different times during development. As a result a well ordered structure of the developing embryo emerges. This process is known as pattern formation. In other words pattern formation in developmental biology, is the mechanism by which initially equivalent cells located at a particular position in a developing tissue of the embryo assume complex forms and functions. Thus, what a cell will become later in the embryo depends on its position within the developing embryo. The final fate of development of a single cell takes place in several progressive steps. For example, during development the pattern formation of the face enables the cells to know where the nose, ears, eyes have to be formed and what muscles are to be placed in the space to support the structure of the face. In animal development, pattern formation is established early in embryogenesis. This patterning during animal development in different organisms is achieved at different times by cellular and molecular mechanisms. The first step in pattern formation is the laying down of the body axes: i) that run from head (anterior) to tail (posterior) and ii) from back (dorsal) to the underside (ventral). Most of the animals that we are taking as examples to discuss development in this course are externally observable as bilaterally symmetrical. This means, that the left and right side of their body are mirror images of each other. The body of such types of animals have an anterio-posterier axis and a dorso- ventral axis. Both these axes are always at right angles to each other and make a coordinate on which the form of the animal is specified (see Fig.10.9). Unit 10 Principles of Development 25 Fig.10.9: The main axes in a developing embryo of Xenopus laevis. Vertebrate animal bodies have externally visible symmetry but the internal organs are not symmetrical for instance the heart is on the left side and liver on the right. These axes in the embryo are established very early in development. Several genes are involved in the establishment of axes in the embryo. Before the externally visible body axes get specified in the embryo the egg also always has a distinct polarity or orientation. At the same time that the axes are being formed in the embryo, the cells in the embryo of triploblastic animals get organized into the three different germ layers namely ectoderm (external layer), mesoderm (middle layer) and endoderm (internal layer).In the case of diploblastic, animals the cells in the embryo get organized into the two different germ layers namely ectoderm (external) and endoderm (internal). The positions of cells in the germinal layers of both diploblastic and triploblastic, embryos of the animals which are laid down during gastrula stage show their prospective fate by the end of development. During further emergence of pattern, the cells of these layers acquire different identities (become different) so that they form the different tissues and organs of the body like skin, muscle, blood cells, neural cells etc. Figure 10.10 gives a diagram of the fate of the three germ layers in animals. Fig 10.10: Generalised cell fates of the three germ layers of triploblastic animals. Block 3 Developmental Biology of Vertebrates-I 26 Pattern formation can take place by two mechanisms: (i) cell to cell interactions also known as inductive mechanisms in which one type of cells induces or instructs other cells to change their behaviour and develop in a different way or to become different types of cell; (ii) morphogenetic mechanisms that act on previously established patterns in order to form the three dimensional tissues and organs. This process involves changes in the location of the cells, without changes in their behaviour. For instance, during gastrulation in frogs, the endoderm and mesoderm move inside the embryo from the surface. This results in the formation of the gut which is a tube within a tube structure. The embryo thus, during development undergoes remarkable changes in appearance or morphology. These changes occur due to extensive cell migration which will also cause changes in the shape of the embryo. In this process some cells undergo programmed cell death as well. We will be studying in greater detail about the mechanisms involved in pattern formation in unit11 and unit 13. 10.5.2 Fate Maps Since the early 1900s, as you will recall, experimental embryologists became interested to find out the fate of each cell in the developing embryo and what it would gave rise to in the adult animal. Initially it was not easy to trace the cell lineage (which structures will be formed from the cell and its descendants at a later stage of normal development) or cell fate of individual embryonic cells. Thus, embryologists used different techniques to label groups of cells in the early stages of embryonic development and followed their progressive development until the stage of a fully formed embryo. By using various labelling techniques of groups of cells from early stages to end of the embryo formation, embryologists were able to construct fate maps or diagrams of areas of the embryo for depicting the fate of the various cells during normal development. Most embryonic cells have predictable fates. Cells in different embryos of same species will develop same structures or will have the same fate. In fact, most early vertebrate embryos show remarkable similarities in their fate maps as can be seen in Figure 10.11 Fate maps can be constructed using different methods which are becoming more sophisticated with increasing technological advances. Each has its advantages and disadvantages. TECHNIQUES USED FOR PREPARATION OF FATE MAPS 1) Observing living embryos: some species have embryos that have relatively small number of cells that are transparent and the development of blastomeres can be observed directly under the microscope or the cells may be naturally coloured (pigmented) so that the development can be traced following the coloured cells. E. G. Conklin in early 1900s was able to follow the development of early cells in a tunicate Styela partita. From his studies it was found that only one pair of blastomeres at the 8 cell stage (the posterior vegetal bastomeres) were capable of forming tail muscles. These had yellow pigment hence it was easy to follow their development. Removal of one such blastomere (B4-1) resulted in an embryo with no tail confirming Conklin's fate map of the tunicaste Styla partita. Unit 10 Principles of Development 27 Fig.10.11: Fate maps of some vertebrate embryos in dorsal view (that is looking down on the embryo to see its back view). You can observe that there are general similarities in all. The cells that will form notochord are in the central position. Ectoderm precursors are anterior, mesoderm lateral and endoderm precursors lie posterior to the cells that make the notochord. The dashed lines show the path where the cells will move inside the embryo. 2) Use of Dye marking: vital dyes (dyes that do not damage the embryo but just stain the cells) are often used to trace cell lineages. Walter Vogt in 1929 used such a dye to follow the development of stained cells in newt embryos. However vital dyes get diluted over the period of cell division and become difficult to trace. 3) Fluorescent dyes and radioactive marking: are used to overcome the problem seen with vital dyes. Fluorescent dyes can be used that are so intense that they can be traced for a large number of cell divisions. Similarly radioactive labelling of dividing cells can also help to trace the path of the dividing embryonic cells. 3) Genetic labelling: This is a method of permanently labelling cells in order to follow their path in development. The best method is the formation of chimeras that are embryos created by fusion of cells from two different, but closely related species. The best example seen is of a chick quail chimera made by grafting quail embryonic cells in a chick embryo while it is still in the egg (Fig.10.12). When the chick hatches it also has quail cells in the tissue which develops from the grafted quail embryonic cells. Another method is by using genetic markers. In this case a marker gene is inserted in the nucleus. Each time the cell divides the tagged gene will also duplicate and in this way these cells that express the genetically modified gene can be traced to the cells of the embryo where the gene was inserted. Block 3 Developmental Biology of Vertebrates-I 28 Fig.10.12: Genetic labelling of cells of the living embryos for determining their fate and for constructing the fate map. Cells from the quail embryo are transplanted on chick embryo. As the chick embryo develops the quail cells can be traced in it. Fluorescent antibody: have been used to study development in the transparent, free living nematode (roundworm) Caenorhabditis elegans. C. elegans has been used as a model organism to follow cell lineage from zygote to the adult form under the light microscope by using a fluorescent antibody which is specific for the nematode. The intestine develops from the cells that arise exclusively from one of the four cells that are formed from the first cleavage furrow in the zygote. Other methods that have been used for constructing fate maps, was by destroying particular cells or cell groups by means of a laser beam. Subsequently, the complete fate map and cell lineage of C. elegans has been worked out. The fate map of Drosophila melanogaster has also been studied extensively and has revealed the molecular and genetic mechanisms involved in body segmentation and axis formation (Fig.10.13) Fig.10.13: Fate map of Drosophila melanogaster showing the regions from where the future body tissues and organs will arise. (modified from https://bastiani.biology.utah.edu/courses/3230/db%20lecture/lectures/b11flydv.html Thus, we find that fate maps are essential for understanding the development and formation of structures in the embryo. As newer techniques develop, the accuracy of fate map improved and embryologists have been able to follow individual cells to see what they eventually give rise to. SAQ 3 What is a fate map? Write a few sentences on the usefulness of fate mapping. Unit 10 Principles of Development 29 10.6 CELL DETERMINATION AND DIFFERENTIATION In the earlier section we have seen that fate maps show the areas of embryo that will produce different tissues and organs in the adult and cell fate describes what a cell will become in the future during the normal course of development. We have also learnt that the fate of a particular cell can be traced by labelling it and subsequently observing what structure it will become a part of. The developmental potential or the potency of a cell describes the range of different cell types that it can give rise to. The zygote and very early blastomeres formed in it by cleavage are totipotent. This means that each of the very early blastomere of the zygote has the potential to develop into a complete organism as was demonstrated by experiments conducted by Dreisch (Refer again to Fig 10.4). Totipotency is not seen commonly after the first few divisions in the blastula. As development proceeds, the developmental potential of individual cells decreases until their fate is "determined". This means that each cell becomes committed to form a part of a specific structure and so its fate is determined for following a particular path of development. Differentiation on the other hand, is the process during which the cells stop dividing and acquire the unique structure and functional properties of a particular cell type. This is thus, the last stage in the process when an undifferentiated cell undergoes a series of events to become a specialized cell type like a muscle cell or nerve cell or skin cell etc. 10.6.1 Cell Determination You are aware that blood cells differ vastly in morphology and function from each other and from other specialised cells like the muscle cells, though all of them arise from the same germ layer namely, mesoderm (refer to Fig 10.10 again). However, before these differences arise there is a period when these cells in the mesoderm do not look different from their neighbouring cells despite the fact that their fate has been already determined. This means that they are committed to follow a specific path of development to form particular types of cells The process of commitment takes place in two steps: a) Specification: The fate of the cell is known to have been specified when it is capable of differentiating autonomously even if it is placed in an environment that is neutral like a culture medium in a petri dish (Fig.10.14 a). However, at this stage the commitment of the cell is flexible and it can be influenced by its environment to become another type of cell rather than what it was specified or fated to be (see Fig.10.14 b). b) Determination: this step is after specification of the cell to form a particular type of cell. In the stage of determination of the cell the fate of the cell becomes inflexibly or irreversibly specified or determined to form a particular type of cell. As a result, the cell will still autonomously differentiate into its original specified fate and its differentiation into a Block 3 Developmental Biology of Vertebrates-I 30 particular type of cell will be independent of its environment or its position in the embryo it (see Fig.10.14 c). Fig.10.14: Two differently positioned cells that are fated to form muscle and neuron cells have been taken and isolated from the blastula and grown in a petri dish. a) blastomeres that are specified for muscle cells differentiate into muscle cells and those specified for neuronal cells differentiate into neurons; b) If the blastomere specified but not determined for forming muscle cells is placed in a cluster of neuronal tissue it differentiates into neural cells; c) if the blastomere is already in the determined phase then it will differentiate into muscle cells even if it is placed with neuronal tissue. Specification Embryos of different animal species show different strategies of specification A. Autonomous specification: in this type of specification the blastomeres of the early embryo have a set of critical determination factors called cytoplasmic determinants that they get from the egg cytoplasm. The cytoplasm is not homogenous but has different regions containing cytoplasmic determinants that are often transcription factors or mRNA encoding factors that will influence the cell's development. There is asymmetric segregation of cellular determinants during cell division. Thus, some cells will have more or less of the transcription factors and some may have none. This will result in daughter cells with different cell fates. These cells already "know" what they are fated to become. Some of the best examples of autonomous specification are seen in the development of molluscs, annelids and tunicate embryos (Fig.10.15). In the tunicate embryo experimental results showed that when the blastomeres were separated at the 8 cell stage each blastomere gave rise to the respective cell type it was fated to make. Moreover, if some of the cytoplasmic determinants like those for muscle development were removed and placed in another cell that cell began to differentiate into muscle cells. Such embryos that have their fates specified early in cleavage are known as mosaic embryos and their development is known as mosaic development. Unit 10 Principles of Development 31 Fig.10.15: Autonomous specification seen in tunicate embryo. The cells were separated at the 8 cell stage and each cell gave rise to only those structures that it would have made in the entire embryo. B. Conditional specification: in this process the cells achieve their final fate by interacting with other cells or their local environment. These cells -- to cell interactions may be of various kinds (which we will discuss in greater detail in the next unit). The fate of a cell in this type of specification depends on its position in the embryo. For example, if the cells from a region of a vertebrate blastula 'A' that are known to give rise to the dorsal region of the embryo are taken out and transplanted to the region of the blastula 'B' that gives rise to ventral region in the embryo, then A type of cells will change their fate and differentiate into B type cells. Moreover the region from where A type cells were taken will also continue to develop normally (Fig.10.16). Various experiments conducted by embryologists since the 1890s that dealt with the separation of early blastomeres in sea urchin which resulted in each blastomere giving rise to a separate embryo (recall both Roux's and Dreisch's experiments). Thus, it was seen that each blastomere or cell acquires its identity based on its position and its interaction with its neighbouring cells and molecules with which it comes in contact (Roux's experiment) and when the early blastomeres were separated they lost that interaction and were able to develop into separate embryos (Dreisch's experiment).Embryos, especially vertebrate embryos in which the early blastomeres are conditionally specified were traditionally called regulative embryos. With further use of molecular biology in the process of development it has been Block 3 Developmental Biology of Vertebrates-I 32 concluded that both autonomous and conditional specification is seen to occur in the development of most embryos. Fig.10.16: Conditional specification. a) The fate of a cell depends on its position in the embryo; b) the region from where the cell had been removed compensates for the loss and the embryo develops normally. Syncytial specification: is the third strategy which is seen mostly during embryo development of insects and has been demonstrated best in the Drosophila embryo. The cytoplasm in Drosophila egg already has certain factors or maternal determinants distributed in it unequally. For instance, the anterior most part of the egg contains an mRNA called Bicoid mRNA that encodes a protein called Bicoid. The posterior most part of the egg contains an mRNA called Nanos mRNA that encodes a protein called Nanos. After fertilization of the egg, these two mRNAs are translated into their respective proteins in the cytoplasm, contributed by the ovum. During the early cleavage stage the nucleus alone divides and the cytoplasm does not separate or undergo cellularization to form individual cells. This results in formation of the blastoderm (a blastula having the form of a disc of cells on top of the yolk) which in Drosophila appears in the form of a large cell with many nuclei and a common plasma membrane (Fig.10.17). A cytoplasm that contains many nuclei is called a syncytium as you may recall. It is in this syncytial blastoderm that the cell identities are determined even before individual cells are formed. Research has shown that the concentration of Bicoid protein is highest in the anterior part of the cytoplam of the embryo and declines toward the posterior end. While the concentration of the Nanos protein is maximum in the posterior part of cytoplasm of embryo and declines as it diffuses anteriorly. Thus, the long axis of the Drosophila egg is spanned by two opposing gradients---one of Bicoid protein coming from the anterior side, and one of Nanos protein coming from the posterior side. The Bicoid and Nanos proteins form a coordinate system based on their ratios, such that each region of the embryo will be distinguished by a different ratio or concentration of the two proteins. As the nuclei divide and enter different regions of the egg cytoplasm, they will be instructed by these ratios as to which position should they occupy along the anterior-posterior axis of the animal. Those nuclei in regions containing high amounts of Bicoid and little Nanos will be instructed to activate those genes necessary for producing the head. Those nuclei in regions with slightly less Bicoid but with a small amount of Nanos will be instructed to activate those genes that generate the thorax. Those nuclei in regions that Unit 10 Principles of Development 33 have little or no Bicoid and plenty of Nanos will be instructed to form the abdominal structures. After the 13th cleavage division just before gastrulation the cell membranes are laid down to separate the nuclei. The nucleus is able to keep its position in the embryo because of its exposure to the different concentrations of the cytoplasmic determinants and each cell or blastomere after cellularisation would know what part of the embryo it will become. Thus, the specific fate of each cell is determined both autonomously (from cytoplasmic determinants) and conditionally (by interaction with other neighboring cells). Fig.10.17: Syncytial specification in Drosophila melanogaster. The egg already has the Bicoid mRNA concentrated in the anterior part (head region) and the Nanos mRNA in the posterior region (tail part). After fertilisation the cytoplasm of the fertilized egg would contain these two mRNAS which would encode their respective proteins namely Bicoid and Nanos proteins. The noncellularized nuclei present in the embryo are influenced by the ratio of the opposing concentration gradients of Bicoid and Nanos proteins. The three processes of specification of undifferentiated cells in the embryo lead them to different pathways of cell differentiation. The fate of determined cells becomes self- perpetuating which means that their progeny will have the same fate. In the next sub-section we shall look at the mechanisms involved in differentiation of determined cells. SAQ4 Fill in the blanks i) Differential acquisition of certain cytoplasmic molecules present in the egg result in...................... specification. ii) In...................... development cells cannot change their fate if a blastomere is lost. iii) Capacity for......................development results in the change in a cell's fate if it is transplanted elsewhere in the embryo. iv) Specification by interaction of cells is known as............................ Block 3 Developmental Biology of Vertebrates-I 34 v)...................... specification is seen predominantly in insects where after cellularisation both autonomous and......................specification are involved in...................... of cell fate. vi) As embryonic development proceeds, cells become restricted to a particular pathway they are said to be....................... vii) Most cells physically change once they take their mature form, they are....................... 10.6.2 Cell Differentiation Cytological studies in the early 20th century established the fact that all the somatic cell arising from the fertilized egg contained the same chromosomal complement. This fundamental concept is known as genomic equivalence. In this section we will learn how this concept has been proved to be true by the help of some path breaking experiments. This concept of genomic equivalence has raised several questions. For instance if all the cells contained the same genomic complement then how do cells become different from each other? Furthermore, why do only some cells like the red blood cells (RBC) make haemoglobin proteins which are never produced in other cells of the body? Or why insulin hormone is produced and secreted only from certain cells of the pancreas and never in the kidneys or in the brain? By 1960s, based on experimental evidence, the concept of differential gene expression came into existence to provide answer to these and several other questions and to explain how similar looking cells with the same type of genetic complement differentiate into different types of cells. Genomic Equivalence The best test of whether all somatic cells contain the same complement of genes as the fertilized egg from which they have arisen is to check if the nuclei of the differentiated somatic cells still have the ability or potency to generate all types of cells. If indeed this is the case then a nucleus taken from one type of cell in the body should be totipotent (having the ability to produce all types of cells) and if transplanted into an activated enucleated (nucleus removed) cell should be able to give rise to all the cells of the body. In the 1950s, Robert Briggs and Thomas King conceptualized and conducted the experiments for determining the totipotentency of the nucleus of early embryonic cells.In their experiment they first combined the technique of enucleation and activation of an oocyte of the leopard frog Rana pipens. The oocyte was activated by pricking it with a sterile needle which provided it with the necessary stimulus to undergo all the cytoplasmic and biochemical rearrangements associated with fertilization, including the completion of second meiotic division at the animal pole of the cell. Puncturing the oocyte at this point let the spindle and chromosomes flow out of the cell (enucleation). The nucleus from a donor cell was then removed and by using a micropipette Unit 10 Principles of Development 35 was inserted into this activated, enucleated cell. Briggs and King demonstrated that blastula (early stage embryo) nuclei when transferred into activated enucleated oocytes could direct the development of a completely formed tadpole (Fig.10.18). They also found that while the nuclei at the blastula stage were totipotent, there was a dramatic decrease in the nuclear potency of cells at later stages. For instance, when nuclei from somatic cells of the tail bud region of tadpoles were used in a similar experiment, normal development did not occur. Thus it was concluded that nucleus from developing embryonic cells appeared to lose the ability to direct development as they underwent determination and differentiation. Further work continued using nuclear transplantation studies and in 1962 John Gurdon a PhD student in Cambridge University showed that if the nucleus of a fertilised frog egg was replaced with a nucleus from the cell of the tadpole intestine, the egg could develop into a new frog. Though the success rate was low, it proved that the nucleus of a mature cell still contained the genetic information needed to build all cell types. This was a major landmark in animal development though the acknowledgment of the importance of his work came much later. It was 40 years after his first experiments with nuclear transformation that the Nobel Prize was awarded in 2012 to Gurdon along with Shinya Yamanake, whose lab induced pluripotent cells. Fig.10.18: Procedure used in enucleation and transplantation of a nucleus from a somatic cell in Rana pipens. Nuclear transplantation techniques were used by experimental embryologists to create clones of several species over the years. In 1997 Ian Wilmut and his colleagues demonstrated that somatic cells still had the potency to produce all the cells of the body. The result was Dolly (Fig.10.19), a sheep that was cloned by using the nucleus taken from the mammary gland of an adult sheep and transplanting it into the egg taken from another strain of sheep. This was done in a culture and later the eggs containing the transplanted nucleus were implanted into the uteri of pregnant sheep. Of the 434 sheep oocytes only one survived to become Dolly. DNA analysis however, confirmed that Dolly's cells had indeed been derived from the donor nucleus. This was the final proof of Fig.10.19: Cloned Sheep Dolly Block 3 Developmental Biology of Vertebrates-I 36 genomic equivalence of somatic cells and that the genome is conserved during differentiation of cells. Since the first cloning experiments, cloning of adult mammals has been done in mice, rats, guinea pigs, rabbits, dog, cat, cows and horses. Figure 10.20 shows the cloning of rats using two different types of rats by using similar techniques that were used for cloning Dolly Fig.10.20: Cloning of mice by employing similar techniques that were used to clone the sheep of Dolly. Unit 10 Principles of Development 37 BOX 10.1 You would certainly have heard about stem cells and their use in medical research. Cell mechanisms of determination and differentiation are the basis of this research. Stem cells refer to cells present in embryos and adult that retain their ability to differentiate into many kinds of cells. In humans, stem cells have been found in bone marrow, brain, in some muscles, skin and liver. These cells can normally differentiate into a limited number of cell types. Because of this they are referred to as multipotent cells and not totipotent. Under normal conditions our bodies use the stem cells to regenerate and replace tissue such as skin, blood, liver etc. Stem cell research hopes to exploit the multipotent characteristics of cell in order to regenerate damaged and diseased tissue and organs. Research is going on with some success in stem cell therapy to regrow damaged spinal cord tissue, replace diseased heart muscle tissue and skin tissue and in search of cures for cancer which is really a disease of abnormal cell division and differentiation patterns. 10.6.3 Differential Gene Expression Genomic equivalence established the general principle that somatic cell nucleus of an organism contains the complete genome established at the time of fertilization and there is no change in the genetic content after differentiation of cells. If all cells in the developing embryo have the same genetic content, differences between cells must be due to different gene activity in each cell type. This means that only a small portion of the genome is expressed or "turned on" in each cell. As a result only those RNAs are synthesized in the cell that will translate (make) only those proteins that will provide structural information to these cells to help them differentiate and ultimately develop into very different cell types. It is important to understand how various signals cause cells to express different portions of their genetic information. Nuclear transplantation and cell fusion experiments reveal that gene activity is controlled by the cytoplasmic environment. New cytoplasmic signals can activate previously silent genes and silence previously active genes. Such genetic control continues throughout embryonic development and results in the generation of a variety of cell types with different gene activities. As the zygote cleaves, its cytoplasmic contents contributed by the egg do not pass uniformly into different blastomeres. The descendent cells at cleavage, therefore, have different cytoplasmic environments. This initiates a pattern of embryonic cells with a distinct programme for gene expression. Later in embryonic development, interactions between blastomeres release new signals, which then determine additional group of cells to activate new sets of genes. The first evidence for differential gene expression came from the study of polytene chromosomes of Drosophila larvae (Fig.10.21 a). In polytene chromosomes DNA duplication occurs in many rounds without any division and separation of the cell. The cell size increases and so as a result, the many stranded DNA is easy to observe. It was seen that the banding pattern of the chromosomes was identical throughout the various cells of the larva. No loss or addition of any chromosome portion was seen in different cell types in the larva. However, different parts of the chromosome were 'puffed up' in different Gene expression is the process by which the genetic code - the nucleotide sequence - of a gene is used to direct protein synthesis and produce the structures of the cell. Genes that code for amino acid sequences are known as 'structural genes'. Block 3 Developmental Biology of Vertebrates-I 38 cell types suggesting that different RNA was being synthesized in different cells and while one part was puffed up in one cell type the other genes were silent and so not puffed up in that cell (Fig.10.21 b). Fig 10.21: Polytene chromosome from salivary gland of Drosophila malanogaster. http://www.biologydiscussion.com/chromosomes/useful-notes-onchromosomes/34691 By the late 1980s it was understood that gene expression could be regulated basically at 4 steps and this could explain the mechanism of differential gene expression. i) At the stage of gene transcription so that only those genes that are required for that particular cell type are expressed in the form of nuclear RNA. Transcription is the first step of gene expression and its control involves general and tissue specific transcriptional regulators. ii) At the nuclear RNA processing stage, by regulating which part of the RNA or which of the transcribed RNAs are allowed to leave the nucleus. iii) At the RNA translation stages by regulating which of the mRNAs are to be translated into proteins. iv) At the protein modification stage, so that only those proteins are modified that can provide the structure and function to the specific cell type. In order to understand the way genes are expressed it would help if you were to read Box 10.2 so that you can quickly recapitulate the principles of the central dogma of biology. Before proceeding further let us look at the structure of a gene. You know that a gene is a distinct sequence of the DNA molecule that has the information to make a polypeptide or a nucleotide. Figure 10.22 shows the gene responsible for making the β-globin in the haemoglobin molecule. The β-globin is made up of different components. Only the parts labelled exons will provide the information for the appropriate amino acid sequences for translating the appropriate protein and not the parts labelled intron. On this portion of DNA the transcription will start at the transcription initiation site and end at transcription termination site. This Unit 10 Principles of Development 39 segment will form the HnRNA (hetrogenous n RNA) and will undergo processing in which the transcribed introns will be spliced or removed. This would the result in putting together the consecutive exons and forming the mRNA. In further modification, 7-methylguanosine (G) cap and a poly (A) tail will be added to the 5-prime end and 3- prime end of this mRNA respectively. This modified mRNA will then leave the nucleus to move into the cytoplasm where it will be translated into a protein of interest. Look at the gene again in the figure 10.22. You will see that the DNA has a portion marked promoter which is not transcribed. The promoter is very important as it is at this location where the proteins and factors bind which can thus regulate or enable the process of transcription. Furthermore in the upstream (anterior end) region or in some cases even in downstream (posterior end) region and also in the introns, there will be sequences that will be able to influence the promoter to initiate transcription. These sequences are regulatory factors also known as enhancers (that stimulate), repressors or silencers (that inhibit transcription). Fig.10.22: The making of beta-globin for the haemoglobin molecule. Beta-globin is inactive unless it is modified and combined with alpha globin to make the complete haemoglobin molecule. Block 3 Developmental Biology of Vertebrates-I 40 Let us now see how regulation of gene expression can occur. Earlier in the section we had said that gene expression can be regulated at all stages from transcription to the final making of the gene product, namely protein. We will discuss the regulation at the transcription level in a little more detail so that you can understand this important mechanism. If a gene is to be regulated then first it should be accessible to a variety of factors that can bind to the DNA and regulate its expression. 1) The first step would be the loosening of the chromosome so that it can change from hetrochromatin state (when the DNA is packaged and coiled around the histones) to the euchromantin state (when the uncoiling of the DNA occurs) so that the gene becomes accessible to a variety of factors. Therefore, regulating the chromatin to uncoil or coil will influence where and when in the genome certain genes can be expressed. For this process small organic molecules can be added or removed from the histones around which the DNA remains coiled. DNA remains coiled if the histones have methyl groups attached to their tails and uncoiling occurs when the methyl groups are replaced by acetyl groups on the histone tails. It is a general rule that acetylation will give access to the promoter region and initiate active transcription, while methylation will repress transcription. Experiments have shown that this is one way of regulating gene expression. 2) Another way of regulation is by transcription factors that bind to the promoter region and can turn genes on or off. If even one transcription factor is changed in the embryo it can have dramatic effects. For example, the loss of a gene ultrabiothorax can have drastic effects in insects. This gene is a homeobox gene which contains a transcription factor, whose absence changes the segmentation pattern in the fly. As a result the fly forms two pairs of wings instead of the usual one pair due to loss of a gene ultrabiothorax. There are different transcription factors which bind to their affiliated DNA binding domains and these are very specific. Let us take the example of an enhancer region on a gene. A transcription factor specific to that enhancer forms a complex with it and can change the shape of DNA so that the promoter and transcription factor- enhancer complex come near the promoter. This causes a very important enzyme DNA polymerase to bind to the promoter which causes the transcription of a particular RNA. If transcription factor binds to the repressor region it will stop or slow down the gene from expressing. For example, the human foetal embryo liver cells synthesize serum albumin but only after a certain stage of development. Till then the gene that encodes for serum albumin is silent or repressed as a transcription factor is bound to the repressor domain. Thus, again it is the specific combination of the transcription factor with the enhancer or repressor that will regulate the rate of transcription in specific cells and cause differential gene expression. Often there is a cascade of reactions in which the gene for making a transcription factor is regulated by another transcription factor. Therefore, it is important for you to understand that there are a variety of ways to regulate the gene and the protein it forms, which in turn Unit 10 Principles of Development 41 influence the differentiation of the incredible varieties of cells seen. Scientists have still a lot to learn and discover about these mechanisms. BOX 10.2: Central Dogma of Biology Central dogma is the sequence of events that enable the information stored in the DNA of the nucleus to be copied and transcribed into instructions for translating proteins in the cytoplasm of the cell. These proteins decide the ultimate structure and function of the differentiated cell. The sequence is given below in the chart. DNA Transcription (double stranded opens up and is copied) HnRNA (single stranded transcript containing exons and introns) Processing (noncoding regions or introns removed to form consecutive coding region or exons) Transport (leaves the nucleus and enters the cytoplasm) Ribosome --mRNA complex (amino acid polymerisation via tRNA) Polypeptide Assembly Modification (includes folding, addition of functional moieties such as carbohydrates,phosphates or cholesterol groups) Functional Protein (supports structure and functional properties of the cell). CHAPTER 11 In Unit 10 we had discussed how descriptive embryology evolved into the discipline of developmental biology and how fate maps in early animal development help to trace the development of differentiated cells. We learnt that genes control development by controlling when and where proteins will be synthesized. This differential gene expression is the cause of determination of different cell fates and cell differentiation. In this unit we will look at early development processes through which the embryo acquires its shape and structure. The differentiated cell types are not placed in the embryo in a random manner but are arranged in organized structures for example limbs, heart, lungs, eyes wings and other internal organs. This formation of organized structures from simple epithelial sheets and mesenchymal masses is termed morphogenesis. The early germ layers- ectoderm, endoderm and mesoderm undergo extensive rearrangement, through regional specification and directed movement of cells from one location to another in the embryo to form the three dimensional animal body. These morphogenetic processes involve cell shape changes, cell migrations and cell to cell interactions which will determine how the embryo will get its shape. We will learn more about cell to cell interaction and patterning with the example of the development of a Block 3 Developmental Biology of Vertebrates-I 46 vertebrate eye and the influence of chemical signals called morphogens in the process. Finally we will look at the different processes of cell death which are important in morphogenesis for giving rise to the shape and contours of organs in the embryo. ObjectivesObjectives Objectives Objectives After studying this unit you should be able to: discuss the mechanism of differential gene expression; describe the basic mechanism of cell movements and cell migrations in morphogenesis; explain cells adherence and its role in morphogenesis; describe the different types of cell signalling and their role in morphogenesis; explain how pattern formation occurs through morphogens and inductive signal; and discuss the different mechanisms of cell death and their need in development. 11.2 MORPHOGENETIC PROCESSES Before we start discussing the morphogenetic processes, it is important to know that all embryonic cells are basically of two types - epithelial and mesenchymal (Fig.11.1).This categorisation of embryonic cells relates to cell shapes and cell behaviour rather than to their embryonic origin. Epithelial cells can arise from all three germ layers and mesenchyme arises from mesoderm and ectoderm. An epithelium is a sheet of cells that rests on a basement membrane and each cell joins its neighbour by specialised junctions; the cells have a distinct apical -- basal polarity. Epithelial cells form sheets, tubes and lining of organs. Mesenchyme is made up of loose cells embedded in the extracellular matrix lying between the ectoderm and endoderm of the developing embryo. It fills up much of the embryo and later forms the fibroblasts, adipose tissue, smooth muscle and skeletal tissues. Fig.11.1: Epithelial cells and mesenchymal cells are the two basic cell types in the embryo. Unit 11 Cell to Cell Communication 47 Remember that for the embryo to form its structure from a single cell the zygote, the processes that take place broadly are cell division to make more cells; then these cells have to be differentiated as per cell fate; the cells have to move, rearrange themselves, change their shapes and aggregate to form different tissues and organs. Thus the embryo acquires a recognisable shape and structure of the particular organism. All this takes place because of communications that occur between the cells of the embryo. Till a little more than two decades ago, not much was understood regarding the manner in which cells communicate with each other to construct an organism from a single cell, the fertilized egg or zygote, through genetically controlled events. However, towards the late 20th century it became clear that molecules in or on cell membranes were involved in the ability of cells to adhere, migrate and influence other cells. In this section we will discuss three basic processes that require cell to cell communication through the cell surfaces - cell movement, cell adhesion and cell signalling. These are the key properties of cells that are involved in changes in embryonic form. Let us look at the property of cell motility first. 11.2.1 Cell Movements Cell movements or motility is an active phenomenon that is essential for many biological processes such as morphogenesis, wound healing, immune response and even cancer metastasis. In this unit our focus is on morphogenetic processes where cell movement is targeted to specific sites in the developing embryo to form tissues and organs, A good example of these cell movement or cell migrations is seen in the movement of neural crest cells (multipotent cells that arise from embryonic ectoderm and give rise to different types of cells) and germ cells in vertebrates. Short range movements are also important and cell motility is responsible for both movements of individual cells as well as change of shape while remaining part of a tissue. For example, the folding of epidermal sheets to make tubes is caused by changes in the shape of the cells. All cells move and change shape by rearranging their internal cellular skeleton (cytoskeleton) or scaffolding by contraction of the cytoskeleton fibres made up of microtubules and microfilaments that are actin -- myosin complexes also termed as actomyosin complexes. These actomyosin complexes are simpler version of those seen in muscles. The energy required to produce the movement comes from adenosine triphosphate (ATP). In non muscle cells these actomyosin complexes are concentrated in the region just below the cell membrane. Moving cells also have a polarity that is, a front and a back region. The mechanism of cell movement can best be seen in the movement or crawling of fibroblasts (a type of connective tissue that secretes collagen found in the extracellular matrix) on a substratum which is the extracellular matrix inside the embryo or glass surface of petri-plates under in vitro conditions (Fig.11.2). Fibroblasts extend a flat process called lamellipodium which is rich in microfilaments made up of a crisscross of actin. From the lamellipodium extend focal contacts that attach it to the substratum and these are connected to the microfilament bundles of the lamellipodium. During movement the microfilaments contract and the body of the cell is pulled forwards. Cells of the Block 3 Developmental Biology of Vertebrates-I 48 embryo essentially move in a similar manner. Instead of the large lamellipodium they may have multiple thin filopodia that make the contact with the extracellular matrix as they move over it. In the embryo the cell movement is directional towards a signal which is a chemoattractant that is detected by the proteins on the cell membrane. These chemoattractants are diffusible molecules and the cells move towards increasing concentrations of the diffusible molecules. You will learn more about this phenomenon in a later section. Fig.11.2: Fibroblast moves by extending the large flat lamellipodium that makes contact with the substratum. Cell shape also changes by the contraction of microfilaments and the associated motor proteins actin and myosin. If the constriction happens in the apical region of epithelial cells it will reduce the apical surface area and elongate the cell (Fig.11.3). This happens initially during invagination during the process of gastrulation when the cells leave the epithelium to move inside the gastrula (you will learn more about the cell movements during gastrulation in Unit 12). Unit 11 Cell to Cell Communication 49 Fig. 11.3: Cell shape change in epithelial cells by apical constriction and result in elongation of the cell. SAQ 1 Fill in the blanks: i) Cell movement takes place because of rearrangement of.....................of the cell along with.....................\... molecules. ii) Cells in the embryo move over........................................... iii) Embryonic cell movement is a response to.................................... from other cells. 11.2.2 Cell Adhesion The other important property that is involved in changes in animal embryonic form is cell adhesiveness. Animal cells stick to one another and to intercellular matrix through interactions involving cell surface proteins. These cell surface proteins can determine how specifically and tightly the cells adhere to one another. These proteins can affect the cell surface tension and contribute to the arrangement of cells in the three germ layers and later in different tissues. Differences in cell adhesiveness also help to maintain the boundaries between different cell types and tissues. Different cell types have both, different types and different amounts of cell adhesion molecules on cell surfaces thereby, having selective affinity for each other which is important for giving positional information to embryonic cells. Because of cell adhesion embryonic cells do not sort out randomly but can actively move to create tissue organisation. The differential adhesion interactions of cells form a certain hierarchy. If cell type A is situated internal to cell type B and the final position of cell type B is situated internal to C, then cell type A will always be internal to C. There are different Block 3 Developmental Biology of Vertebrates-I 50 classes of cell adhesion molecules, but the major cell adhesion molecules appears to be cadherins (calcium dependent adhesion molecules). Cadherins are transmembrane proteins that interact with other cadherins present on adjacent cells. Cadherins are anchored to their cell by a complex of proteins called catenins (Fig.11.4). This cadherin- catenin complex is the classic adherin junction seen in epithelial cells. As the catenins bind to the cytoskeleton of the cell they integrate the epithelial cells and keep them together. Cadherins perform several related functions. (i) their external domains serve to adhere cells together. (ii) cadherins link to and help assemble the actin cytoskeleton, thereby, providing the mechanical forces for forming sheets and tubes of cells. (iii) cadherins can serve to initiate and convert signals that can lead to changes in a cell's gene expression Fig.11.4: Diagram showing cadhedrin - cadherin cell adhesion. Inside the cell the cadherin molecule associates with a catenin molecule which itself is bound to the actin microfilament system of the cytoskeleton (adapted from Takeichi 1991). Unit 11 Cell to Cell Communication 51 Cadherins can be of different types (see Box 11.1) and cadherin of one cell binds to the other cells by the same type of cadherin. For example,cells with E-cadherin will stick to other cells with E-cadherin and sort out from cells with N- cadherin. This type of binding is known as homophilic binding. The sorting of cells was first demonstrated in the 1950s. Since it was known that amphibian tissue dissociate into single cells when placed in alkaline solutions, a single cell solution of the three germ layers of amphibian embryos was made and when the pH was restored these cells adhered to one another in the petri dishes. Thus the behaviour of recombined cells could be studied. The results of the experiment were striking , the cells sort out into their cell type and into their own regions (Fig.11.5). Fig 11.5: Reaggregation and sorting of cells from two different amphibian neurulae.Presemptive epidermal cells from a pigmented embryo and neural plate cells from a unpigmented embryo were dissociated and mixed together. The cells first aggregate together and then the cells segregated according to their cell type position. The presumptive epidermal cells cover the neural cells. The sorting of cells is the combined effect of cell adhesiveness and cell movement. Initially the cells move randomly but then they seek similar cells to aggregate because they have stronger adhesive interactions. The ectoderm is the tissue with the strongest cohesive interaction among cells so it forms the outermost layer of the embryo. The less cohesive mesoderm and endoderm cells are arranged internal to the ectoderm. Cell sorting hierarchy is, therefore, strictly dependent on the amount of cadherin interactions amongst cells. In vitro experiments with fibroblasts that are generally migratory cells and do not express E- cadherin, showed that when activated E-cadherin genes were added to the culture, the fibroblasts started expressing E- cadherin and they became tightly bound to each other and started behaving like epithelial cells. Blocking the function of cadherins by using antibodies that inactivate cadherin or by blocking its synthesis at translation stage can prevent epithelial tissue from forming and causes the cells to disaggregate. Block 3 Developmental Biology of Vertebrates-I 52 Cell adhesion, cell movement and formation of epithelial sheets and tubes depend on the ability of cells to attach to extracellular matrix. When epithelia are to be made the attachment has to be very strong but when cells move or migrate, they have to break their attachment to other cells and reform them at another location. In some cases the extracellular matrix has to serve as a pathway for the moving cells, providing direction or the signal for a developmental event. Fibronectin a component of extracellular matrix in a way paves the roads on which the cells move, for example, fibronectin paths lead the germ cells to gonads and heart cells to the midline of the embryo. The binding of the cell to the extracellular matrix takes place through another family of adherin molecules called as integrins and the mechanism is not dependent on calcium unlike that in cadherins. Integrins are large protein molecules that span across the cell membrane. On the outside of the cell, it binds to the fibronectin of the extracellular matrix; on the inside of the cell, it serves as an anchorage site for the actin microfilaments that move the cell. Box 11.1: Cadherin molecules. There are many type of cadherin molecules and cadherin -- cadherin attachments are the strongest when they are of the same kind. E-cadherin: epithelial cadherin is expressed on all early mammalian embryonic cells and later gets restricted to the epithelial cells of embryo and adults P --cadherin: this is placental cadherin expressed on the trophoblast of the placenta and on the uterine wall epithelium. It possibly facilitates the attachment of the placenta to the uterus N- cadherin: is neural cadherin, first seen on mesodermal cells in gastrula cells as they lose their E-cadherin expression. It is also seen on cells of the developing central nervous system. C-cadherin: is found to be critical for keeping the blastomeres together in Xenopus and for normal development for gastrulation cell movements. Protocadherins: are calcium dependent adhesion molecules like cadherins but lack the connection to cytoskeleton through catenins. SAQ 2 a) How are boundaries between different tissues maintained? b) What is the difference between cadherins and integrins? 11.2.3 Cell Signalling Cells typically communicate with each other by use of chemical molecules or signals. These molecular signals are highly diverse and are responsible for specific protein-protein interactions, which can result in diverse cellular responses like changes in gene transcription, cell metabolism, cell migration and cell death. The signal molecules generally contact other proteins that may Unit 11 Cell to Cell Communication 53 be housed in or on the plasma membrane of the target cells. The signal protein molecules are called ligands (a general term for molecules that specifically bind to other molecules). The proteins that are attached to or embedded in the cell membrane of the target cells are known as receptors. A receptor in the membrane of one cell can bind a similar receptor in another cell in homophilic binding. In contrast, heterophilic binding occurs between different receptor types. Binding of a ligand to a receptor generally sets up a signal transduction pathway. The major signal transduction pathways all appear to have a common theme (see Fig.11.6). Each receptor spans the cell membrane and has an extracellular region, a transmembrane region, and an intracellular cytoplasmic region. When a ligand binds to its receptor's extracellular part, it induces a shape change in the receptor's structure. This shape change is transmitted through the membrane and alters the shape of the receptor's cytoplasmic part, giving that domain the ability to activate cytoplasmic proteins. Such a conformational change often makes the cytoplasmic part become enzymatically active, using ATP to phosphorylate specific tyrosine residues of particular proteins. Thus, this type of receptor is often called a receptor tyrosine kinase (rTk). The active receptor can now catalyze reactions that phosphorylate other proteins, and this phosphorylation in turn activates their latent activities. Eventually, the cascade of phosphorylation activates a dormant transcription factor or a set of cytoskeletal proteins. Fig. 11.6: Structure and function of a tyrosine kinase receptor. Signal transduction pathways that end in expression of a gene in the target cell are generally slower than those that enzymatically activate biochemical pathways or regulate cytoskeletal elements for movements. Block 3 Developmental Biology of Vertebrates-I 54 Cell signals can be categorised according to the distance travelled in the body as: autocrine, paracrine, direct contact and endocrine (see Table 11.1). Table 11.1: Types of cell signalling Paracrine signalling Takes place over short distances between cells by diffusion Juxtacrine signalling Takes place between cells in direct contact Autocrine signalling Signals are received by the same cell that sent them or from adjacent cells of the same kind Endocrine signalling Signals are carried over long distances through the blood stream to the target cells Direct signalling Occurs through gap junctions between neighbouring cells. Allows flow of small molecules between cells In an embryo, communication between cells can occur (i) across short distances, such as between two neighbouring cells through their cell membranes in direct contact, or between a cell membrane and extracellular matrix secreted by another cell called juxtacrine signalling (Fig.17.7 A&B), or (ii) between neighbouring cells through the secretion of proteins into the extracellular matrix, called paracrine signalling (Fig.11.7 C). In the next section we shall examine cell to cell interaction using these two types of communications. Fig.11.7: Modes of communication between cells of the embryo. A) Paracrine signalling in which one cell secretes a signalling protein or ligand into the environment and across some distance from many cells. Only those cells can respond that have receptors for that particular ligand. The receptor can respond, either rapidly through chemical reactions in the cytosol, or more slowly through the process of gene and protein expression; B) and C) Juxtacrine signalling is local cell signalling carried out via membrane receptors that bind to proteins in the extracellular matrix (ECM) or directly to receptors from a neighbouring cell. 11.2.4 Epithelial-Mesemchyme Transition All the cell processes that we have learnt in the previous sub-sections are seen to be integrated in another morphogenetic process --the epithelialmesenchyme transition also known as EMT. During this process the Unit 11 Cell to Cell Communication 55 stationary epithelial cells get detached from the basal lamina and change their identity to become migratory mesencymal cells that can invade tissues and form organs in new places in the embryo. EMT is usually initiated by a paracrine signal from neighbouring cells that initiates gene expression in the target cells. This gene expression instructs the target cells to downgrade their cadherins, release their attachment from their basal laminin, rearrange their cytoskeleton components and secrete the extracellular matrix molecules that are characteristic of mesenchymal cells. EMT can take place involving individual cells or the collective epithelial cells (Fig.11.8) Fig.11.8: Epithelial --mesenchyme transition. A) individual epithelial cells can detach and change into mesenchymal cells; B) a sheet of epithelial cells that moves along the front end towards the direction of migration. This EMT is very important in the developmental process. For example, it is seen in the formation of neural crest cells from the dorsal most region of the neural tube in fish, birds and mammals. It is also seen in the formation of chick and mouse mesoderm where cells that were part of the ectoderm detach and convert into mesodermal cells that migrate into the interior of the embryo; and in cells that detach from the somites and migrate around the developing spinal cord to ultimately form the vertebrae. In all these cases the epithelial cells lose Block 3 Developmental Biology of Vertebrates-I 56 their "epithelialness" that is the breakdown of the basal cadherins-intercellular matrix adhesion and cadherin--cadherin junction at the apical region of the cells, thus loss of cell to cell adhesiveness before they begin to behave like mesenchymal cells and migrate. In adults the process of EMT is needed for wound healing, regeneration of tissue and is the cause of metastasis of cancer cells, where the solid tumour cells detach from the tumour and migrate to other parts to form more solid tumours. SAQ 3 a) How would you define a ligand in cell- to cell signalling? b) What is the difference between juxtacrine and paracrine signalling? c) How is EMT used in the embryo and in the adult? 11.3 CELL--CELL INTERACTION In the last section we discussed the role of cell adhesion, cell motility and cell signalling as crucial processes in morphogenesis. We should also realise by now that from the early stages of embryogenesis, cells do not function in isolation or in a random manner. All cell behaviours like cell adhesion, cell migration, differentiation and cell division are regulated by signals being passed from one set of cells to another. These cell to cell interactions allow the embryo to get its form and shape. But how do organs develop in their proper place in the embryo? And how do cells "know" that they have to migrate and position themselves? Pattern formation is the process by which the cells find their positional information. There are two general modes of pattern formation: (i) through use of morphogen gradients and (ii) by sequential induction. Let us first take a look at the role of morphogen gradients. 11.3.1 Morphogen Gradients Pattern formation in the embryo can involve gradient of chemical signals known as morphogens. This term was coined by Allen Turing in 1952 for substances whose distribution through diffusion would determine the development of cell