MBios 401/501: Lecture 14.2 Cell Differentiation I Slide #1 Cell Differentiation Cell Differentiation I -Basic principles of differentiation (p1305-1320) -C-elegans (p1321-1327) Cell Differentiation II -Pattern formation (1328-1341) -Sensory bristles of drosophila (1356-1359) The next two lectures are going to address cell signaling during development. Developmental biology and tissue differentiation are both enormous and complicated topics. In fact there are entire courses that you can take devoted to these subjects, and so you should be aware that this course is going to cover only the most fundamental aspects of development as well as one or two selected topics. I m going to suggest that you concentrate on the material that s actually presented in this and the following lecture to guide you in figuring out what to study. Specifically I m going to cover the topics listed on this slide, although the remainders of chapters 22 and 23 are certainly interesting and worthwhile if you have the time to read them. Slide #2 Cell Differentiation -All of the different cell types in a multicellular organism are derived from a single fertilized egg through differentiation. -Differentiation is the process by which an unspecialized cell becomes specialized. -Differentiation can involve changes in cell size, shape, polarity, metabolic activity, responsiveness to signals, gene expression profiles. -During differentiation certain genes are turned on while other genes become inactivated. -Cells become different from one another because they synthesize different sets of RNA and proteins. -A differentiated cell will thus develop specific structures and perform specific functions distinct from the cell of origin. -Cell differentiation depends on changes in gene expression and not on changes in the nucleotide sequence of cell s genome. The statements on this slide are relatively self evident if you consider that all cells in an organism are derived from a single cell and that all cells have pretty much the same DNA, with only minor exceptions. Therefore, cells differ from each other by virtue of the genes that are actually active in the cell, as well as the presence of specific types of RNA and protein that arise as a consequence of unique genes being activated. The topic of this lecture is really how different cells become that way, given that they all start from a single original cell. Although Page 1 of 10
I m not going to take the time to read these statements to you, you should be familiar with them. Slide #3 All animals have a similar organization -Figure The diversity of animal life on the planet is really astonishing, but for the most part animals all have a very similar general body plan. In its simplest form, an animal is made up of three different layers of tissue, all of which are produced very early in development. These layers form the inside, outside, and middle of the animal, and the names of these tissues in the embryo are the ectoderm (for the outside), the endoderm (which is on the inside), and mesoderm (for everything in the middle). The illustration on this slide shows the embryo of a sea urchin, which at one point was a major model system for studying development, mostly because the embryos are really small and transparent, and it s possible to watch everything going on in it relatively easily. The sea urchin adult really doesn t get much more complicated than the picture shown on this page, but even mammalian embryos like humans start out as a group of cells organized into the three basic layers. Slide #4 Basic mechanisms of development are essentially the same among vertebrates as well as invertebrates. -Homologous proteins are often functionally interchangeable between different species. - ~50% of genes present in C. elegans (nematodes worm), Drosophila (fly), and humans have clearly recognizable homologs in one or both of the other two species. This is especially true for genes that regulate cell interactions and for gene regulation (ie., transcription factors). -Figure 22-2b One of the most amazing aspects of development is that even the molecular components of signaling systems that organize the basic body plan are conserved across a huge range of organisms. In total, almost half of the genes involved in development are highly conserved. This is especially true of signaling processes that regulate early development. The images on this slide show two very good examples. On the left you see a portion of the brain of a mouse that regulates movement called the cerebellum. A mutation in the gene called engrailed results in poor development of this area of the brain. That s shown in this image on the right at the top of that picture. If you introduce a copy of that engrailed gene from an insect into the mouse this will allow that region of the brain to develop normally. Similarly, the images on the right show what happens when you express the gene Pax-6 from mice and squid in an insect. The Pax-6 gene is an early regulator of eye development, and the expression of mammalian and molluscan versions of these genes still creates an eye in the fruit fly, although sometimes in odd places. Page 2 of 10
Slide #5 No title -Figure The images on this slide try to show how early genes in different animals can be the same yet produce widely different end products such as wings on an insect and arms on a mouse or human. The key is that the development of any final structure is really the result of a series of cascading genes that get activated in sequence. In the images on this slide, you can imagine that the gene labeled gene 1 is a master control of a developmental program that will tell tissues to make an appendage, while genes 2 and 3 are more specific as to what kind of appendage is going to get made. Further along there will be other genes that will specify what sorts of proteins will be expressed in each cell in the appendage and so on. So the process gets more specific over time. Slide #6 Cell differentiation precedes morphological changes -Figure Cells generally become differentiated long before the program of differentiation actually results in visible changes to the cell. The process a cell goes through when it becomes destined to become a particular cell type is called determination. Before a cell is determined, it can respond to environmental cues and change the final outcome of its developmental program. Once a cell is determined it will follow a single developmental path, no matter where it s placed in the body. The diagram at the top right of the slide represents the outcome of many different transplantation experiments where tissues were moved from one place to another in early development and take on the characteristics of the destination site. The same experiment conducted later on in development produces a different result, where the cells that are moved retain the developmental program that was present at the initial site or their site of origin. The figure below that diagram shows wing and leg buds of a chicken embryo stained to show the location of transcription factors involved in limb specific gene regulation. Even though the limbs look almost the same, wing buds express Tbx5 but not Tbx4 orpitx1 (you don t have to know the names of those genes but just understand this concept). Even earlier limb buds are still pretty far down along their developmental pathway towards the formation of a final appendage. For example, if you take a small piece of tissue from a leg limb bud and place it on the end of a wing limb bud the result will be a wing that has toes on the end of it. The diagram on the left of the slide shows the experiment schematically. I ve included an image from the original 1955 paper where this experiment was actually done, and it shows a toe at the end of a feathered chicken wing. Page 3 of 10
Slide #7 Cell Specification: How do cells become different? -Although two daughter cells produced from mitosis will each have an identical copy of mother s cell genome, they will often have different cell fates. -by segregating molecules -by exposure to different environment -Figure 22-11 So this slide presents the most fundamental question in developmental biology, which is how can two dissimilar cells be produced from a single? This is a central issue because if this didn t happen, then the division of a fertilized egg for example would just produce more cells that are identical to each other, and that s not what happens. The figure on this slide shows two possible mechanisms. First, it s possible that there is an unequal distribution of some factor that s already been programmed into the cell before it divides, such that one cell get more or less of the factor after the cells divide. In fact, egg cells are generally not just homogeneous masses and they are often produced within the female with different distributions of RNA molecules and other morphogenic factors. The second possibility is that cell responds to different local environmental cues. Many cells use this approach. For example, if a skin stem cell divides, it gives rise to one other stem cell, plus another cell that will go on to differentiate into a mature adult skin cell. The environmental cues that are recognized include things like the attachment of the stem cell to an extracellular matrix molecule, but not the cell that will go on to differentiate. Egg cells also use this strategy. Some examples of environmental cues that may be recognized by eggs include things like light or the site of sperm entry. We will look at examples of this in the following lecture. Once a cell has become segregated in some fashion, the unequal distribution of molecules can then become a determining factor that will leave one cell to adopt a different cell fate than the other. Slide #8 Cell Specification: Also created by positive feedback -Figure 22-12 The small differences between daughter cells that might be created by unequal distributions of morphogenic factors are enforced through feedback systems. An example of this is shown by the diagram on this slide where slight differences are created randomly in the middle rows of cells by cell division, but the cell with more X s then instructs the cell with fewer not to make any more X protein. The result is the fully differentiated pair of cells seen at the bottom of the slide. This is exactly the type of system that plays a role in the differentiation of the neural epithelium that we discussed in the section of the cell signaling lecture covering the Notch/Delta signaling system. This type of developmental regulation generates a binary or an on/off distinction between two cells. Page 4 of 10
Slide #9 A morphogenic gradient of Sonic Hedgehog control limb development in vertebrates -Figure 22-12 Binary distinctions between cells are not the only type of transition that plays a role in development. Morphogenic gradients are another common mechanism used by developing systems as an alternative. Morphogenic gradients allow finely graded or transitional changes from one place of an organism to another. An example of this is shown on this slide. The image on the left is a four day old chicken embryo that was stained to show the expression of a vertebrate hedgehog gene called Sonic hedgehog. During the formation of the limb, a special group of cells at one side of the limb secretes this hedgehog signaling protein. The protein diffuses out from its source and forms a morphogenic gradient which controls the development of the limb bud. It s possible to take a piece of this Sonic hedgehog expressing tissue and place it at other places in the developing embryo and the diagram on this slide shows the result of transferring a piece of this tissue to the other side of the limb bud. The result is a wing that displays a mirror image duplication of the far end of the wing. This is a result of the fact that the tissues of the limb bud are programmed to respond to a gradient of Sonic hedgehog in an appropriate manner. With tissues close to the source forming fingers or toes, and tissues further away forming arm bones. Slide #10 Morphogenic Gradients -Signals from cells outside of group alters developmental/differentiation pathway. -Generally, the signal is limited in time and space so that only a subset of cells take on induced character. -These are longer-range signals that can diffuse through the extracellular medium. -Figure The diagram on this slide shows schematically how a time course of developmental events can proceed using secreted morphogens in which an initial developmental signal affects the development of a localized group of cells. The initial cells that respond to the morphogen are colored black in the middle row of cells on this diagram. After they differentiate they follow the developmental program that involves secreting an additional morphogenic signal. Cells close to that source respond by turning blue, although others further away do not. In this way the single original signal resulted in a structure with three different cell types. This process can be repeated to produce very precise, spatial regulation of developmental events in an embryo. The developing insect larva is an excellent example of this. An original morphogenic gradient of the protein Bicoid, which is shown at the top of the slide on the right, is set up in the egg of a fruit fly. This gradient produces developmental changes leading to the expression of what is called gap genes, which are shown in red, yellow, and green in the second embryo. These cells then produce additional morphogenic gradients, and we will go over this entire mechanism in more Page 5 of 10
detail in the next lecture. Slide #11 Morphogens are short and long-range inducers that can exert graded effects -Figure -Table 22-1 The diagram on the top of the slide shows schematically how developmental patterns like the one shown by the drosophila larva on the previous slide might be generated. Initially two cell types, labeled A and B might be created along a very large gradient of morphogens such as the one produced by the egg polarity gene bicoid on the last slide. The cells labeled B then begin to signal the cells in the region labeled A to differentiate further. The cells produce a new cell type called C. Cell type C also produces signals which then cause A type cells to become D cells and the B type cells to become cells of type E. When we discuss the development of the nematode worm, C. elegans, we will look at specific examples of this type of developmental signaling pattern. The table at the bottom of the slide shows some of the most common signaling elements that are used during development and these include the Wnt, Notch, and Hedgehog signaling pathways that we ve already discussed, as well as numerous growth factors that would operate by interacting with receptor tyrosine kinases. Slide #12 Extracellular inhibitors of signal molecules shape the response to the inducer -Important to limit the action of a signal as well as to produce it -There are antagonists for most developmental signal proteins -act by binding to signal itself or to receptor -Two ways to create morphogen gradients: -Neural development in frog is mediated by inhibiting action of a TGFβ protein, resulting in development of neural tissue in region of highest concentration of inhibitor. TGFβ induces epidermal tissue. -Figure 22-14 The effects of diffusible morphogens need to be limited in both space and time. The limitation can occur by one of two mechanisms. First, a morphogen can just be produced in a restricted area. Cells close to the source receive a high dose of morphogen and others further away receive lower amounts, but if the production or morphogen occurs over a very lengthy amount of time, it s likely that even distal tissues will eventually receive a high concentration of the morphogen. Gradients are often the result of a process that inhibits or destroys the morphogen at more distant sites. Thus there are antagonists for most morphogens. On the right hand side of the slide is an example where an initially uniform expression of the morphogen TGFβ is altered by an inhibitor called chordin, which itself is expressed as a gradient. The result is a TGFβ gradient. Page 6 of 10
Slide #13 Caenorhabditis elegans -Figures The rest of this lecture is going to focus on the early development of the nematode worm called Caenorhabditis elegans, or more commonly C. elegans. C. elegans is a model organism that has been thoroughly studied and its development is fairly well understood. The organism is a small worm that s easily grown in the laboratory, and the adult is transparent enough that every cell in its body can been seen clearly through a microscope. Although it s a fairly simple organism, it still makes muscles, nerves, epithelia, digestive, and reproductive tissues that are homologous to the similar structures found in vertebrates. On the next slide is a time lapse video from the lab of Dr. Bob Goldstein at the University of North Carolina which shows the early development of C. elegans. The movie is pretty informative, up until the embryo develops muscles, at about 7 hours after the first cell division. After that it sort of writhes around so much that the developmental changes are hard to see. So concentrate on appreciating what s going on at earlier time points. Slide #14 Development of C. elegans (courtesy of Dr Bob Goldstein, UNC) -Video No audio Slide #15 In C. elegans, cell lineage is fully known. -Figure 22-18 This slide illustrates one of the most interesting aspects of C. elegans development and is one of the most powerful aspects of this organism as a model system. The organism is simple enough that every cell in its body has a predictable outcome and can be traced directly back to its origin. In all, there are exactly 1031 cells in the adult male worm. The other gender in C. elegans is a hermaphrodite and it has 959 cells. The diagram on this slide is a fate mat showing the development of early cells and also an expanded tree showing the entire development of the digestive system or gut. The lines represent the differentiation of individual cells from precursor cells. If you look at the top of the diagram you can see that even the first cell division gives rise to two cells which have different developmental fates. I ve labeled these branches 1 and 2. The cells that form the entire gut are shown by the blue lines and it is also formed from a single cell that arises at the 8 cell stage of the embryo. In addition, all of the germ cells, or in other words the sperm and egg producing cells, arise from a single cell that arise at the 16 cell stage. We ll Page 7 of 10
look in some detail at that particular cell and its development in the next few slides. Slide #16 Development of germ line producing cells in C elegans -Unequal cell divisions are produced by activity of Par (Partitioning defective) proteins -P granules (green) are segregates into a single cell at each of the first four cell divisions -Figure 22-19 This slide takes a closer look at the developmental signaling pathway that leads to the formation of the germ cell line producing cell that I showed you on the previous slide. The egg cell contains structures called P granules, which contain proteins and RNA molecules that will be used to differentiate the germ line of cells. P granules are prepackaged into the egg during egg cell production by the female, but they are initially randomly distributed. The entry of the sperm into the egg creates a polarity that s used by the cell to redistribute the P granules as well as other developmental factors. This is one example of an egg cell responding to an environmental cue to create differences within itself. The entry of the sperm into the egg is shown on the figure on the slide at the far left. The blue dye is staining DNA and you can see the eggs chromosomes in the center and the sperm s nucleus entering the cell in the upper left of the egg. There s another sperm that you can see which is attached to the outside of the egg, it s also labeled blue. In the first image the egg has not divided, but the green P granules are already found on the side of the cell opposite the site of sperm entry. The reorganization of the eggs content is accomplished by the activity of proteins called Par proteins. Mutations in the Par protein leads to defects in egg cell partitioning, so Par stands for partitioning defective. Homologs of the C. elegans Par protein are found in vertebrates where they also play roles in cell development. The second panel from the left shows the result after one cell division. You can see that all of the P granules ended up in one cell on the right. After two more divisions there are 8 cells and you can count the blue dots in the upper image to confirm that there are really 8 cells. All of the P granules are still found in only one cell, and this process of partitioning the P granules to one cell continues to the 16 cell stage which is shown at the far right. Slide #17 Cells in C elegans embryos can be identified and their progeny determined: -AB: ectoderm -P: Primordial germ cell -a/b: anterior posterior -E: Endoderm (gut) -MS: mesoderm and stomodeum -C: secondary ectoderm -Figure 22-20 The precise determination of the developmental fate of cells in C. elegans was recognized as early as 1899. The fertilized egg is divided into an anterior and posterior by the entry site of the Page 8 of 10
sperm, which marks the front or anterior end of the embryo. The first cell division results in the formation of anterior cell called an AB cell and a posterior cell called P, which you ll recall from the last slide has the P granules that will eventually determine which cell becomes germ cells. During the next round of cell division the AB cell divides into an anterior and posterior cell designated ab a for anterior and ab p for posterior. While the first primordial germ cell, which was the P cell, divides to form a cell designated EMS (which no longer has any P granules) and a second P cell (that once again has all the P granules) that s called P 2. At the third round of cell division the ab a cell and the ab p cell both divide vertically to now produce four different AB cells. The AB cell lineage of cells will eventually become the outer layer of cells or the ectoderm, and this also defines the axis of the embryo. At the same time, the P 2 cell divides horizontally and the upper daughter cell becomes a C cell, which gives rise to skin, muscle, and nerve cells. The lower cell retains the P granules and becomes a P 3 cell. The EMS cell divides into two additional cells, one labeled E which will become the endoderm and gut, and the other called MS which will produce muscle and mouth parts. Slide #18 No title -The posterior P2 cell uses the Notch signaling pathway to send a signal to Abp cell. Aba cell does not receive a signal & therefore doesn t respond. -P2 produces Delta which binds to Notch receptor on Abp cell and to activate Notch-responsive genes. -P2 also produces Wnt, which binds to Frizzled receptor on EMS cell. Wnt signal polarizes EMS cell, controlling orientation of mitotic spindle. EMS cell then divides to generate two daughter cells, one of which becomes future gut founder cell and one of which becomes committed to form muscle and other body parts. -Figure 22-21 This slide shows the developmental signals that are involved in producing the Abp, MS, and E cells. These are the ones that I actually want you to kind of know. The fate of these cells are all determined by signals that come from the P2 cell which contains P granules. P2 produces Delta, which activates notch receptor on the Abp cell. This causes it to become different from the Aba cell and ends up determining the anterior posterior axis of the embryo in much the same way that bicoid distinguishes the axis of the Drosophila embryo. The P2 cell also produces a member of the Wnt family of signaling proteins, which is recognized by the EMS cell. The site of that cell closest to P2 becomes the E cell and then the embryonic gut, while the side further away from the P2 cell becomes the MS cell. Slide #19 Selected cells die by apoptosis as part of the program of development and differentiation. -The control of cell numbers in development depends both on cell division and on cell death. -C. elegans generates 1031 somatic cell nuclei; 131 of these die by apoptosis. -Genetic screens have identified three genes that are crucial for apoptosis: ced-3, ced-4 (cell death abnormal) and egl-1 (egg laying defective protein-1). Ced-9 represses apoptosis. Page 9 of 10
-ced-3 encodes a caspase homolog -ced-4 homolog is Apaf-1 -egl-1 homolog is Bad -ced-9 is Bcl-2 -Figure 22-23 Finally, programmed cell death also plays a role in the development of C. elegans. Exactly 131 cells undergo programmed cell death during development and they undergo apoptosis under the control of proteins homologous to the proteins I introduced on the lectures on apoptosis, including the caspases and the Bcl-2 family of proteins. In C. elegans, most of these proteins are called ced proteins, which stands for cell death abnormal. Page 10 of 10