Assignment 1: Introduction to Developmental Biology PDF

🏛️ Assignment 1: Introduction Dev. Biology Class date @February 10, 2025 Course 🧬 Developmental Genetics Status 4-see class Exams ⭕ Ex...

🏛️ Assignment 1: Introduction Dev. Biology Class date @February 10, 2025 Course 🧬 Developmental Genetics Status 4-see class Exams ⭕ Exam Dev. Gen. Test date February 25, 2025 Week day Lecture https://hstalks.com/t/4095/developmental-biology/? Materials biosci Lecturer Cheryll Tickle Overview of development biology and its history Developmental biology is the study of how a single cell, the fertilized egg, gives rise to a new organism. In just over seven weeks, a fertilized human egg, gives rise to an embryo with many recognizable features. For example, the head with the eye can clearly be seen as can the arms and the legs. Developmental biology, while focusing on embryonic development, also covers events that occur after birth, postnatally, such as growth and aging. Developmental mechanisms are also involved in regeneration, which is the ability of an adult organism to replace missing parts. As development is fundamental to evolution, evolutionary developmental biology, sometimes called evo-devo is a very active area of research. Why study developmental biology? Assignment 1: Introduction Dev. Biology 1  It is intrinsically interesting topic how an organism develops is its own right. In addition, knowledge about developmental mechanisms has applications in medicine and agriculture. With regard to medical applications, we would like to understand why development sometimes goes wrong. Perhaps, less obviously, developmental biology can also help our understanding of diseases such as cancer. This is because cancer employs the same cellular mechanisms as development and even the same molecules. Another clinical area in which developmental biology is having an increasing impact is in regenerative medicine. If we understand how cells and tissues are used to build organs in the embryo, this could suggest new approaches to repair and replacement of damaged or diseased tissues. Branches of developmental biology: Developmental biology has its roots in embryology and anatomy, but it involves other branches of biology; from genetics and genomics, molecular biology and biochemistry through to cell biology, mathematical biology and biophysics can also make important contributions. Model organisms in the developmental biology landscape Most of our knowledge about developmental mechanisms comes from studying the embryos of model organisms. Eggs easily available and the embryos readily manipulated Some of these, chickens and amphibians such as frogs and newts, were widely studied by experimental embryologists because their eggs are easily available and the embryos readily manipulated. Facilitate the study of the genetics of development Others fruit flies, mice, thale cress (arabidopsis), zebrafish, and nematode worms have come to the fore because they facilitate the study of the genetics of development. As we shall see, some of the key advances in understanding general principles that can be applied even to human development have come from Assignment 1: Introduction Dev. Biology 2  studying flies and worms. Identification of genes in model organisms controlling the body plan Discovery of the organizer A key advancement that laid one of the foundations for developmental biology was the discovery of the organizer, a small group of cells in the early amphibian embryo. This group of cells controls the laying down of the main body axis. When the organizer is transplanted to the opposite side of another early embryo, cell-cell interactions between the transplanted organizer and the host tissue leads to the host tissue making a second body axis. Twinned tadpole A twinned tadpole resulting from an organizer transplant → Cell-cell interactions occur frequently during development and how cells tell other cells what to do is a central issue. First clues of genetic basis of development It marks the birth of developmental biology. These clues came from studying mutants of the fruit fly, Drosophila. In some of these mutants, one part of the body is replaced by another. Assignment 1: Introduction Dev. Biology 3  On the left, is the normal fly. A pair of antennae have developed between the two eyes. In contrast, in the mutant fly on the right, legs have developed instead of antennae. Through identifying the genes involved in these type of changes in the body plan (Hox genes → a subset of homeobox genes, are a group of related genes that specify regions of the body plan of an embryo along the head-tail axis of animals), a complex of genes was discovered, which specifies the regional identity of the segments of the fly body and determines the type of appendages they develop. It was a very big surprise that even though our body doesn't look anything like that of a fly, vertebrates, including humans, have related complexes of genes, which seem to operate in the same way as the complex in the fly and serve a similar function. Other genes discovered through analysis of Drosophila mutants turn out to have vertebra counterparts, including genes that function in cell-cell interactions. Developmental biology and clinical genetics The importance of identifying developmental genes is that they provide a direct link to clinical genetics. When genes affected in human patients in which development had gone wrong began to be identified, it was very satisfying to see the two fields (fruit fly mutants + genes responsible for human conditions) converge as the genes are the same. The mouse as a genetic model for development At the same time, as the advances in genetics in the fruit fly, the mouse was beginning to be established as a mammalian genetic model for development. The breakthrough was identifying how to fertilize mouse Assignment 1: Introduction Dev. Biology 4  eggs in vitro, maintain the earliest stages of development in culture, and then, successfully implant the embryos into a surrogate mother and obtain live births. This work provided the foundation for subsequently establishing in vitro fertilization in humans, which can be used to treat infertility. In the developmental biology context, it also provided a way of studying the earliest stages in mouse development, which normally takes place hidden in the mother. At about three days after fertilization, a blastocyst is formed with an internal cavity, and a small group of cells at one side of the cavity can be seen. This group of cells is known as the inner cell mass. It's from this group of cells that the embryo forms. A key discovery was that the cells of the inner cell mass are pluripotent embryonic stem cells. These embryonic stem cells can give rise to all the different cells of the body. They also provided important tools for making knockout mice test gene function. They can be grown in culture and genetically manipulated, and then introduced into a host blastocyst to generate a transgenic mouse from which knockout mice can be bred. It was for this work that Cappechi, Evans, and Smithies were awarded the Nobel Prize. More recently, gene- editing techniques have been developed, which make these types of experiments much easier, as the components required to edit genes can be injected directly into the fertilized egg, and more sophisticated experiments can be made including, experiments in which more than one gene can be functionally inactivated at the same time. Stem cells The defining feature of stem cells such as embryonic stem cells is that when a stem cell divides, one of the daughter cells may differentiate into a particular cell type, while the other daughter cell may become another stem cell. Assignment 1: Introduction Dev. Biology 5  This means that populations of stem cells are self-renewing. Stem cells are not only found in embryos, but also in adult tissues that continually renew themselves such as the skin, the lining of the gut, and the blood. They're also found in tissues that can regenerate such as muscle and even in certain regions of the brain. Unlike embryonic stem cells, however, adult stem cells can only give rise to a limited number of different cell types. New opportunities from genomics - 2000s The most recent advance that has impacted developmental biology as it has all of biology is genomics. The ability to sequence whole genomes has led to new ways of analysis and also, the opportunity to examine non-coding regions of the genome such as regulatory sequences of DNA. The genomes of all model organisms studied in developmental biology have now been sequenced. Genomics is also having an increasing impact on evo- devo studies, because we can readily sequence the genomes of non-model organisms and, for instance, study the evolution of genes and their regulation. Human developmental genetics and cellular processes How does a fertilized egg give rise to a new organism? Assignment 1: Introduction Dev. Biology 6  We will take the human embryo, again, as our example and look at the human life cycle. The first stages are similar to those we have seen in the mouse, and lead to the formation of a blastocyst that implants in the uterus. Soon after implantation, the crucial stage takes place in which the body plan is laid down. This crucial stage is known as gastrulation and it takes place in a sheet of cells derived from the inner cell mass. It involves extensive rearrangements of cells to generate the three main layers of the body and also the main body axis is laid down. Lewis Wolpert has said that, "Gastrulation is the most important event in your life- more important than birth, marriage, or death." In the human embryo, as we have seen in the amphibian, there is an organizer that controls gastrulation. In mammalian embryos, this organizer is called a node. After gastrulation, considerable changes take place in the shape of the embryo involving the folding of various tissues. For example, a tube is formed along the back of the embryo which is the forerunner of the central nervous system. The next stages in development between four and eight weeks involve organogenesis - the formation of organs. One of the first organ systems to develop is the vascular system as this is required to nourish the growing embryo. Assignment 1: Introduction Dev. Biology 7  As we have seen by about eight weeks, all the parts of the body have been laid down, and the embryo is now known as a fetus. The remainder of the period before birth involves maturation of the organs and extensive growth, and this growth continues after birth. Transcription and translation All the information to make a new organism is encoded in the DNA of the genes. This information can be transcribed into RNA which is then translated into proteins, and it is the proteins that a cell makes that determine its activities. Cell activities turn genotype into phenotype during development. Cells have a relatively modest repertoire of cell activities. They can proliferate, make more cells, or they can die. They can change shape, move, and migrate. They can stick to other cells or they can lose their attachment to other cells. They can also interact with other cells by producing and responding to molecular signals. Developmental processes in which cell activity is involved Cell activities are employed in four major developmental processes that are involved in making an embryo. Growth is when a single cell gives rise to many cells. Morphogenesis is the process that shapes the embryo and its organs. Cell differentiation is the process that ultimately produces specialized cells for different functions. Assignment 1: Introduction Dev. Biology 8  Pattern formation is the process that controls the spatial arrangement of differentiated cells and tissues and generates anatomy. In development, these processes act in concert but we will now consider each of them in turn. Growth of the human embryo and foetus Growth is mainly the result of cell proliferation, outstripping cell death leading to an increase in size. A considerable amount of growth takes place even before birth. Growth is controlled by both extrinsic and intrinsic factors, and is probably the least understood developmental process. We need to understand, for example, how the different parts of the body grow and how their growth is coordinated. What ensures that growth ceases when the correct size is achieved, and why do some organisms keep on growing? Morphogenesis Development involves not only getting bigger but also changing shape. This series of drawings by the French embryologist Tredern shows the growth of the chick leg, and how individual toes are formed. In fig 14, the digits can be seen to be joined by a web of tissue, and it's now known that localized cell death is responsible for their separation. The genetic control of cell death was discovered about 30 years ago by studies on the development of the nematode worm, and Assignment 1: Introduction Dev. Biology 9  similar genetic control of cell death is found in vertebrates. Another important cell activity involved in shaping the embryo is the ability of cells to change shape. This computer simulation shows how changes in the shape of cells in one region of a cell sheet can lead to the bending of the sheet. This mechanism, together with cells moving past each other, is employed in making the neural tube the forerunner of the central nervous system. Computer modeling of this sort is often used to provide insights into morphogenesis. Cell migration Cell migration is a key activity in gastrulation. A gene encoding a fluorescent protein has been introduced into cells at random so that the behavior of the cells expressing this protein can be tracked. Trajectory of the moving cells. These type of imaging techniques have revolutionized the way in which cells can be observed in living embryos and are widely used to study morphogenesis at the single-cell level. Cell differentiation Cell differentiation has been likened to playing a jukebox because it involves cells playing different tunes from the playlist, in other words, differential gene expression. Differential gene expression and pattern formation Gene expression can be controlled in several different ways. Localization of cytoplasmic determinants & asymmetric cell divisions Assignment 1: Introduction Dev. Biology 10  Cells that inherit different cytoplasmic determinants when they divide, and this makes the two daughter cells different. This kind of division is known as an asymmetric cell division, and it's common in development. Extracellular factors Another way in which cells become different is by being exposed to extracellular signals that act as differentiation factors. There is no detailed knowledge about how genes are switched on and off at the molecular level. Master genes encoding transcription factors This involves proteins known as transcription factors that bind to regulatory regions in the DNA that control the expression of particular genes. Some genes, known as master genes, encode transcription factors that act as major switches in cell differentiation, and lead to a cascade of expression of other genes. When master genes or other transcription factors that are lineage-specific are introduced into fibroblasts, they can convert the fibroblasts into the appropriate differentiated cell type. Heritability of differentiated cell state - epigenetics Another important issue about cell differentiation is heritability. This is important so that cells maintain their integrity. This involves epigenetic changes that do not affect the gene sequences, but involve modifications in the chromatin that can be passed on to daughter cells. Nuclei of differentiated cells can support development A critical experiment in frogs showed that cell differentiation involves differential gene expression, and that the nuclei of differentiated cells can still support the development of an egg. When a nucleus from a cultured adult skin cell or tadpole (larval stage of an amphibian) gut epithelial cell is transferred into an unfertilized egg whose own nucleus has been destroyed, the egg undergoes development and produces a tadpole. The tadpole is a clone of the animal from which the nucleus was taken. In other words, it is genetically identical to that animal. Cloning has also been carried out successfully in mammals, most famously producing Dolly The Sheep. The important feature of this experiment is that it Assignment 1: Introduction Dev. Biology 11  shows that the pattern of gene activity in the nucleus of a differentiated cell can be reversed when it's exposed to a different cytoplasmic environment. In this case, the cytoplasm of the unfertilized egg. Cellular reprogramming The experiments on frog embryos we have just considered, together with the finding that introducing master genes can dictate the differentiated cell types, have led to a landmark discovery that adult skin fibroblasts can be reprogrammed to pluripotent stem cells by introducing genes that encode embryonic stem cell-specific transcription factors. This work was carried out by Yamanaka, and the cells that are produced in this way are known as induced pluripotent stem cells. The importance of this finding is that such cells could be made from the cells of a patient, and then used to generate cells that repair diseased or damaged tissue by differentiating them into the appropriate cell type. Another potential route reducing patient-specific cells is through the direct reprogramming of cells from the patient, using master genes or lineage-specific transcription factors, and introducing them into the fibroblasts. These strategies are being explored for use in regenerative medicine. Pattern formation Assignment 1: Introduction Dev. Biology 12  An important issue about cell differentiation during development is how it is spatially organized. This is accomplished in the process known as pattern formation. There are several ways in which differentiated cells and tissues become spatially organized. Reaction-diffusion (Turing-type mechanism) Reaction-diffusion spontaneously generates a pre-pattern with peaks and troughs of concentration of a morphogen. This mechanism for making patterns is also known as a turing type mechanism, after the mathematician, Alan Turing, who first discovered it. Such a pre- pattern can produce repetitive patterns, not just stripes and spots. Positional information In contrast, in positional information, a mechanism put forward by Lewis Wolpert, there is no pre-pattern. But, instead, cells are informed of their position, and then interpret its positional information to differentiate in an appropriate manner. Sorting out This involves cells differentiating at random, and then moving to the appropriate position. Clock-like (timing) mechanisms Timing mechanisms- timing when cells differentiate into a particular cell type, and produce patterns. Wolpert’s French flag model In contrast to reaction-diffusion mechanisms in which the stripes are all the same, with positional information, you can make stripes of different colors, and Lewis Wolpert illustrated vividly this ability to make stripes of different colors by considering how a line of cells will differentiate to form the pattern of the French flag. Cell position in the line is defined by a concentration gradient of a morphogen. Cells at different positions in the line are exposed to different concentrations of the morphogen, and cells then interpret this positional information to differentiate into either a blue, or a white, or a red cell. Assignment 1: Introduction Dev. Biology 13  Chick wigs development The developing chick wing has been one of the main models for investigating the process of pattern formation. The wing develops from a small bud of cells that grows out from the body wall. As it does so, cells differentiate to form the skeleton, shown here in black. Parts of the skeleton along the main axis of the wing are laid down in sequence, with the digits forming last. The chick wing has three digits that form a pattern. A pattern of three digits, traditionally labeled 2, 3, and 4. Each digit is morphologically distinct. So, what are the mechanisms that produce this pattern, and ensure that the digits develop in their proper positions? The answer should help us understand why a thumb develops at one edge of our hand and a little finger at the other. Assignment 1: Introduction Dev. Biology 14  Cell-cell interactions in the chick wing bud Classical embryological experiments carried out about 70 years ago identify two sets of cell-cell interactions in the developing wing bud. One set of interactions involved the apical ectodermal ridge. A sheet of cells that covers the tip of the limb bud, and controls outgrowth. The other set of interactions involves a small group of cells inside the limb bud, known as the polarizing region. The polarizing region acts as an organizer to pattern the digits. The apical ridge and the polarizing region interact, so that pattern formation is linked to outgrowth. The polarizing region was discovered by a grafting experiment in chick wing buds. When a group of cells from the lower edge of the wing bud was grafted on the opposite side of a second wing bud, this had a dramatic effect. Six digits developed instead of three, and the additional digits were in mirror-image symmetry with a normal set. Assignment 1: Introduction Dev. Biology 15  Morphogen gradient model for the chick wing bud: The effect of the polarizing region graft can be explained in terms of a French flag model. The proposal is, that the polarizing region produces a morphogen (chemical substance that influences the cellular development) that sets up a concentration gradient across the wing bud. Cells at different positions would be exposed to different concentrations of the morphogen, and this would inform them of their position. They, then, use this information to make the appropriate digit. Digit 4 at high concentrations, and digit 2 at low concentrations. Following a polarizing region graft, a U-shaped gradient of the morphogen results and hence, the mirror image symmetrical pattern of digits. closer to the polarizing region → high concentration of morphogen (blue color from the French flag - digit 4) away to the polarizing region → less concentration of morphogen (red color from the French flag - digit 2) Universality of polarizing region morphogen: The morphogen from the polarizing region is universal. Tissue from the equivalent region of a mouse limb bud can induce additional digits in the chick wing. The morphogen is the same in the mouse and chick, but the interpretation of positional information depends on the responding cells- that the additional digits are chick wing digits. Morphogens and the polarizing region in limb development Assignment 1: Introduction Dev. Biology 16  What is the morphogen? It is now known that the secreted protein encoded by the sonic hedgehog gene (Shh) is the polarizing region morphogen. The sonic hedgehog gene is a vertebrate gene related to the hedgehog gene first identified as a developmental gene in drosophila. The larva of the fly mutant have many crystals and look prickly, hence the name. Sonic hedgehog is expressed in the polarizing region of the chick wing, and a gradient of sonic hedgehog protein has been found across the wing bud. In addition, beads soaked in sonic hedgehog protein, can induce a mirror image pattern of the digits just like the polarizing region. Sonic hedgehog is not only involved in limb development, it's also involved in the development of many other organs in the embryo. Abnormal sonic hedgehog activity has also been found in several different types of tumors. As we would expect, the sonic hedgehog gene is also expressed in the polarizing region of mouse limb buds. In mouse embryos in which sonic hedgehog is functionally inactivated, the development of structures below the elbow or the knee are very reduced. At best, only a single digit-like structure develops. The failure of digits to develop is because sonic hedgehog controls the growth of the limb bud, both directly and indirectly via maintaining the apical ectodermal ridge. Digits 4 and 5 of mouse limb come from the polarizing region Unlike the chick wing, the mouse limb has five digits. Genetic experiments in which the fate of the polarizing region cells have been traced, show that digits 4 and 5 in the mouse limb, come from the polarizing region itself as shown by this blue staining. Cells that form digit 5 remain longer in the polarizing region than cells that form digit 4. Assignment 1: Introduction Dev. Biology 17  Comparison of digit pattern formation in chick wing and mouse limb Therefore, it appears that in the mammalian limb, digits 1, 2, and 3 are specified by the concentration of sonic hedgehog just like the three digits of the chick wing. But, digits 4 and 5, are specified by the timing mechanism and are linked to the length of time that cells express sonic hedgehog. Self-organization of a digit pre-pattern The limb also has a remarkable capacity for self-organization. Assignment 1: Introduction Dev. Biology 18  When a chick leg bud is disaggregated into single cells which are then mixed together and reaggregated again to form a recombinant limb bud, digit-like structures develop, although all of these look the same ⇒ identity of each digit depends of additional information. This suggests that there is a pre-pattern in the developing limb generated by reaction-diffusion, and that it is the identity of each digit that is specified by positional information or a timing mechanism. Several different mechanisms of pattern formation appear to operate together to produce the pattern of digits. In the reaction-diffusion model that helps explain this type of self- organization, there are typically two types of morphogens interacting: 1. Activator (locally active morphogen): This is a substance that promotes its own production and often stimulates the production of the second morphogen, the inhibitor. The activator acts locally and triggers a response in nearby cells, which can lead to the formation of specific structures like digits. 2. Inhibitor (diffused morphogen): This substance spreads or diffuses through the surrounding tissue. Its role is to prevent the activator from acting over too wide an area. This diffusion creates periodic peaks and valleys of morphogen concentration, which generates the regular pattern necessary for the formation of digits. Assignment 1: Introduction Dev. Biology 19  A cis-regulatory DNA sequence controls expression of the Sonic hedgehog gene in the limb The discovery has given new insights into both clinical medicine, and into evolution. This sequence is about 1 megabase away from the sonic hedgehog gene, and is an example of long-range control of gene expression. The sequence was identified in a mouse mutant called sasquatch in which a transgene had accidentally inserted into the sequence. Sasquatch has an additional digit in the limb as shown by the star, and the sonic hedgehog gene is expressed both in the normal polarizing region, and also at the opposite side of the limb bud. Mutations in Shh limb regulatory sequence in human patients and domestic animals Mutations in this regulatory sequence have subsequently been found in patients with an additional digit. This provides an explanation of how this digit arose. Mutations have also been found in domestic animals such as cats and chickens. The cats are sometimes referred to as Hemingway cats, as there is a famous tribe of cats with additional digits at Hemingway's old home in Assignment 1: Introduction Dev. Biology 20  the Florida Keys. This tribe of cats is descended from a cat that belonged to Hemingway. The silkie chicken also has additional toes. Mutations in Shh limb regulatory sequence in snakes Mutations in the sonic hedgehog limb regulatory region have also been found in snakes. Although in this case, the mutations lead to loss of sonic hedgehog expression in the polarizing region, rather than expression at both sides of the limb bud. ⇒ mutations do not lead to Shh in both sides of limb buds, but instead, there is a loss of expression of Shh in polarizing region, which affects the limb development. When the regulatory region is deleted in mice, using gene-editing techniques, sonic hedgehog expression is lost in the limb bud and the limbs are severely truncated resembling limbs sonic hedgehog knockout mouse embryos. In some snakes such as pythons, hind limb buds form but do not develop. Replacing the mouse sequence with the sequence from pythons and cobras, shows that the sequence in these animals (pythons and cobras) is non-functional. The regulatory sequence from snakes cannot drive sonic hedgehog expression in the limb bud, and severely truncated limbs develop. This study suggests that mutations in the regulatory sequence of Shh in snakes were an important factor in the loss of limbs during their evolution. The inability to properly express Shh in the limb buds resulted in truncated limbs or their complete loss, as observed in most species of snakes today. This provides an explanation of how hind limbs might have been lost during snake evolution. In contrast, the human regulatory sequence and the regulatory sequence from the coelacanth, and both function normally as replacements for the mouse sequence. Environmental agents that affect limb development We have just discussed an example in which the genetic basis for a change in human limbs has been discovered. But, only around 50 percent of Assignment 1: Introduction Dev. Biology 21  changes in limb development in humans are thought to have a genetic basis. Environmental agents such as chemicals and infectious agents known as teratogens, can also interfere with development. The best-known teratogen that affects limb development is the drug thalidomide. Patients whose mother took the drug to treat morning sickness between 20 and 36 days of development, were affected. Experiments with chick embryos have shown that thalidomide prevents the growth of blood vessels into the limb buds. This leads to cell death and affects the laying down of structures along the axis of the limb. Final comments: As we have seen from this brief introduction, developmental biology is a mature discipline and the general principles have been elucidated. However, a lot of detail is still lacking, though there is much to do. The main areas that look likely to spearhead future research include evolutionary developmental biology, which has been given new impetus by advances in genomics. Also, ecological developmental biology looks as though it may be a growth area. This is the effects of the environment on developmental processes which would also include teratogenesis. Another major focus emerging is on human development. With the use of 3D cultures of human cells, for example, to study organogenesis. Finally, stem cells, in particular, human stem cells, are being intensively studied as they have tremendous potential in regenerative medicine. Assignment 1: Introduction Dev. Biology 22