Unit 10 Principles of Development PDF

UNIT 10 PRINCIPLES OF DEVELOPMENT Structure 10.1 Introduction 10.5 Emergence of Patterns Objectives Pattern Formation Fate Maps 10.2 Developmental Stages...

UNIT 10 PRINCIPLES OF DEVELOPMENT Structure 10.1 Introduction 10.5 Emergence of Patterns Objectives Pattern Formation Fate Maps 10.2 Developmental Stages Common to all Animals 10.6 Cell Determination and Differentiation 10.3 Historical Background of Developmental Biology Cell Determination 10.4 Animal Models in Cell Differentiation Developmental Studies Differential Gene Expression Classical Models: Sea Urchin and Frog 10.7 Summary A later Model: Drosophila 10.8 Terminal Questions Newer Models: C elegans and 10.9 Answers Danio 10.1 INTRODUCTION 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. 9 Block 3 Developmental Biology of Vertebrates-I 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. 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. 10 During the course of development, the developing cells of the zygote Unit 10 Principles of Development 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. 11 Block 3 Developmental Biology of Vertebrates-I 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 12 organ in the embryo at the beginning of development. Unit 10 Principles of Development 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 Fig.10.2: Pre the first to see the blastoderm of the chick which is the clear, yolk free formation theory germinal disc, also called the blastodisc and is in the form of a single layer of depicted by embryonic epithelial tissue and gives rise to the chick embryo. William Harvey homunculus. 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: 13 Block 3 Developmental Biology of Vertebrates-I 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. 14 Unit 10 Principles of Development 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. 15 Block 3 Developmental Biology of Vertebrates-I 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 16 Unit 10 Principles of Development 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 17 Block 3 Developmental Biology of Vertebrates-I 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 18 iv) of having a short life cycle, Unit 10 Principles of Development 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: Fig. 10.6: Sea urchin egg seen under the 1. Gametes can be obtained easily and artificial spawning and fertilization microscope with and rearing is possible. sperms surrounding it, for fertilization. 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. 19 Block 3 Developmental Biology of Vertebrates-I 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 20 studies of reptiles and birds as well as mammals. Developmental studies of Unit 10 Principles of Development 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 21 Block 3 Developmental Biology of Vertebrates-I 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 22 Unit 10 Principles of Development 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 (a) (b) 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? 23 Block 3 Developmental Biology of Vertebrates-I 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 24 coordinate on which the form of the animal is specified (see Fig.10.9). Unit 10 Principles of Development 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. 25 Block 3 Developmental Biology of Vertebrates-I 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. 26 Unit 10 Principles of Development 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. 27 Block 3 Developmental Biology of Vertebrates-I 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. 28 Unit 10 Principles of Development 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 29 Block 3 Developmental Biology of Vertebrates-I 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. 30 Unit 10 Principles of Development 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 31 Block 3 Developmental Biology of Vertebrates-I 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 32 Unit 10 Principles of Development 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. SAQ 4 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 ………………………. 33 Block 3 Developmental Biology of Vertebrates-I 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 34 Unit 10 Principles of Development 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 Fig.10.19: Cloned the cells of the body. The result was Dolly (Fig.10.19), a sheep that was Sheep Dolly 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 35 Block 3 Developmental Biology of Vertebrates-I 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 36 clone the sheep of Dolly. Unit 10 Principles of Development 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 Gene expression is of cells. If all cells in the developing embryo have the same genetic content, the process by which differences between cells must be due to different gene activity in each cell the genetic code - the nucleotide sequence - type. This means that only a small portion of the genome is expressed or of a gene is used to “turned on” in each cell. As a result only those RNAs are synthesized in the direct protein cell that will translate (make) only those proteins that will provide structural synthesis and information to these cells to help them differentiate and ultimately develop into produce the structures of the cell. very different cell types. It is important to understand how various signals Genes that code for cause cells to express different portions of their genetic information. Nuclear amino acid transplantation and cell fusion experiments reveal that gene activity is sequences are known controlled by the cytoplasmic environment. New cytoplasmic signals can as ‘structural genes’. 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 37 Block 3 Developmental Biology of Vertebrates-I 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-on- chromosomes/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 38 transcription initiation site and end at transcription termination site. This Unit 10 Principles of Development 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. 39 Block 3 Developmental Biology of Vertebrates-I 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 40 Unit 10 Principles of Development 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) SAQ 5 a) Fill in the blanks with appropriate word/words in the following sentences i) Cell differentiation is the result of ………………………. expression. ii) During gene expression methylation of histone tails results in …………………… while acetylation causes ……………………. iii) …………………… ……………………bind to enhancer regions of the gene to form a complex that binds to …………………….… to initiate transcription. b) Define genomic equivalence. c) Give one word for each statement given below: i) The zygote cell has the potential to form any cell in the body and so it is …………………… ii) Stem cells have the ability to form a limited variety of cells and so they are …………………… 41 Block 3 Developmental Biology of Vertebrates-I 10.7 SUMMARY After studying this unit you have learnt: The study of embryonic development started more than 2000 years ago. Aristotle put forward the idea of epigenesis which states that embryos were not completely preformed in the egg but structures emerged gradually as the embryo developed. This idea was questioned in the 17th and 18th century and the idea of preformation of the embryo in the eggs or sperm was advocated. However, with the emergence of the cell theory the idea of preformation was laid to rest. As more experimental methods and sophisticated microscopes came into use experimental embryology developed further. Some early experiments in embryology showed that in sea urchin embryos if one of the blastomeres is destroyed the other blastomere develops in half of the embryo and if the blastomeres are separated at 2 cell or even 4 cell, stage, each can develop into a full but small embryo. This was evidence that showed that cells are able to regulate development. Direct evidence of cells being regulated by other cells in the embryo came in 1924 by the experiment of Spemann and Mangold that showed the existence of an organiser region in the early embryo. The role of genes in controlling the development of embryos has only been established in the last 50 years and developmental biology as a discipline was established through inputs from other disciplines such as cell biology, genetics, biochemistry and foremost molecular biology. Development starts with the fertilisation of the egg by a sperm and the resultant zygote undergoes cleavage; followed by emergence of pattern; change in cell form and determination of cell fate; cell differentiation and growth. Pattern formation involves laying down of the overall body plan around the body axes and establishment of germ layers. The concept of germ layers and fate maps of the embryo are useful to distinguish between regions of early embryo that will give rise to distinct cell types and the future tissues and organs of the organism. The developmental potential of early embryo cells is greater than their general fate and as development progresses cells lose their potency and cell fate becomes specified and determined. This means that the internal state of cells changes and they become committed to a specific cell fate. During specification embryos of different species can show any or a combination of three strategies-autonomous (mosaic), conditional (regulative) and syncytical. At this stage the cell’s fate is still flexible and it can be induced to differentiate into another cell type. However once determined the cell has a stable internal change and its fate becomes fixed. All cells of the embryo and the adult organism have the same genome (genomic equivalence) as the zygote. This genomic equivalence was proved by nuclear transplantation experiments involving enucleated cells 42 Unit 10 Principles of Development and transplanting nucleus from other early embryonic cells and later from differentiated somatic cells. The first successful experiment in a mammal was the creation of a sheep named Dolly. Cell differentiation involves the gradual emergence of cell types that have a clear cut identity in the adult, such as nerve cells, red blood cell, muscle cells etc. Initially the embryonic cells fated to become different cell types, differ from each other in the pattern of gene activity that forms different proteins. Over successive cell generation these cell acquire their new structural features and their potential fate becomes more restricted. The central feature of cell differentiation is differential gene expression. This is regulated primarily at the first step of gene expression by transcription factors that bind to enhancers or repressors of gene expression. Transcription factors can be general or tissue specific. However, protein synthesis can be regulated even after the transcription at the successive stages of gene expression. 10.8 TERMINAL QUESTIONS 1. What were the three classical experiments that laid the foundation of experimental embryology? 2. What characteristics should a good model organism have to be useful in developmental biology studies? 3. What is the difference between cell specification, determination and differentiation? Explain with the help of an example in each case. 4. Describe how developmental fate maps can be made? 5. List at least three stages in gene expression that can be regulated to result in differentiated cell types? Explain any one of them with the help of an example. 10.9 ANSWERS Self-Assessment Questions 1. a) Embryology is the science which studies the stages from fertilisation to birth of an organism while developmental biology includes stages of embryogenesis, and growth after birth as well as regeneration and senescence. b) The two proposed views on development –1.epigenesis which says that development happens progressively at every stage and 2. preformation which states that the entire organism is already pre formed in miniture state in the egg or sperm. He emphasized epigenesis a view which is also subscribed by modern animal developmental biology. 2. a) Chick, sea urchin and frog because of the ease of manipulation, large size of eggs, abundance of eggs and ease of rearing. b) Drosophila 43 Block 3 Developmental Biology of Vertebrates-I 3. Fate maps are formed or drawn of those regions in the early embryo that will form the future tissue and organs during normal development. They are significant because they give general information of the cell fates or what cell types they will differentiate into. They are true for all members of the species for which they are drawn. 4. i) autonomous ii) mosaic iii) regulative iv) conditional specification v) syncytial; conditional; determination vi) determined v) differentiated 5. a) i) differential gene. ii) coiling of DNA around nucleosomes; uncoiling /opening of the gene. iii) Transcription factors; promoter region b) Every blastomere, of blastula and every cell of the body, possesses identical genes both qualitatively and quantitatively. c) i) Totipotent ii) multipotent Terminal Questions 1. Refer to Section 10.3. 2. Refer to Section 10.4. 3. Cell specification is the stage when the undifferentiated cells are not committed to their fate and are still flexible to take the fate of another cell type if induced to do so.