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Lecture 07

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Biological Sciences
Ian Brown

Slide 3, Differentiation of Muscle and the Liver: This is a cross section of an embryo in the body region, (diagram of this section) we see the neural tube, the notochord and the somites that differentiate into the muscles of the embryo. At higher magnification, the top shows the neural tube, notochord and somites. The somite is a mesodermal cell clustered around a central cavity. Dermatome; Forms the connective tissue (CT) layer of the skin. Sclerotome; Surrounds the notochord and the spinal cord and becomes cartilage. Myotome; Becomes muscles. This area grows down until they touch. The length of the body differentiates into the different types of muscles in the body. In vivo, in the living embryo we can study this way. Or in vitro, we can grow them in medium in a dish. When you take the cells out of their environment, they undergo de-differentiation, they lose their ability to differentiate because they no longer have the factors available to them to start the process. This does not happen with muscle cells, they are self-inducing with differentiation. Slide 3, Why is the muscle differentiation a good model system?: Three (3) reasons; 1. associated with distinct series of morphological and molecular stages we can recognize 2. muscle specific proteins, such as actin and myocin, turn on during the differentiation of muscle cells and give muscles their contractile ability 3. self-stimulating differentiating system, immature cells can differentiate into mature without any external inducing factors. Slide 4, In vitro Growth of Myoblasts: Embryonic chicken tissue is used. A piece is removed from the thigh and contains myoblasts, these are undifferentiated muscle cells. These are used and mixed with trypsin. This is an enzyme that degrades the tissue proteins (so they won’t stick together) and releases the single myoblast cells. The mixture is spun and a pellet is formed with the single myoblast cells. Myoblasts are put into a dish with medium and they start to divide. This how muscle cells are studied in vitro. Each myoblast has a single nucleus. They go through a number of steps that change the myoblast into differentiated muscle cells. Slide 5, Steps of the Myoblasts: This occurs in the tissue dish without external factors. 1 1. myblast cells undergo cell divisions for several days, these are simple cells with a single nucleus. This results in the cells covering the bottom of the dish, which is called a monolayer. 2. After the monolayer has been formed they stop dividing. Cells control their own division and they turn off division. 3. Myblasts change to multinucleated cells (big cells with lots of nuclei) this is as a result of cell fusion. 4. cell fusion is a trigger for changes in gene expression, this turns off genes in cell division, cell can no longer divide. Other genes are turned on, those for actin and myosin. 5. we observe an increase in muscle specific proteins (actin and myosin) 6. muscle proteins form into muscle fibers which are inside the cells, when they appear the fibers are not doing anything, 7. however a day or two later these fibers within the cells begin to twitch Slide 6, Multinucleated cells are formed by cell fusion; We know this because of two (2) theories: 1. Nuclear division 2. Cell division DNA replication without separation of the daughter cell; Slide 7, Support for Cell Fusion Theory: Experiments that have been done; They investigate the time period in which multinucleated cells form, they form in 6-12 hours. This time period is too short to get many rounds of nuclei within a single cell. No mitotic figures are seen. If two (2) daughter nuclei separate, we can see chromosomes forming, this is mitotic figures. This is not seen, therefore, the normal DNA replication of daughter cells is not happening. When multinucleated cells are forming, we put a DNA synthesis inhibitor, which stops the DNA replication and more nuclei, even in the presence of this inhibitor the multinucleated cells are still formed, therefore, DNA replication is not necessary. We can use time-lapse cinematography, if we film the whole thing under a microscope, we can observe myoblast fusing together. Cell mixing experiments can be used to prove multinucleatic cells form by cell fusion. We can isolate myoblasts from a rat thigh muscle and grow these cells in the presence of a DNA precursor, when they are actively dividing if thymidine is added it will be incorporated into the DNA and the nuclei will be radioactive and they will be seen. This is a DNA marker. Some have their DNA labeled and some do not, if we put the two together in a dish and the cell fusion event occurs. We then see giant multinucleated cells that have labeled and unlabeled nuclei. Therefore, these cells are formed by fusion. We get a mixed nuclei fusion which proves the cell fusion event. 2 Slide 8, Fusion of Myoblasts of Different Species: We can mix myoblasts from myoblasts of different species of animals. Example, rat thigh and DNA precursor, therefore they are labeled. Then add thigh cells from a rabbit, which are not labeled. When they are mixed we get giant multinucleated cells which are a labeled and unlabeled mixture. If we use rat heart cells with rat thigh cells, we do not get giant multi cells because these two types of cells cannot fuse. They must be myoblasts. They can even be from different species. Slide 9, How do we examine changes in myosin mRNA profile? The genes involved in cell division are turned off and muscle specific proteins are turned on. How do we know this occurs? How do we determine the stage when the genes are turned on? We did Northern Blots to determine this stage. This can also be done by in situ hybridization, this allows us to see the stage where the genes are turned on. Northern Blot (NB): We need to make a cDNA probe in able to detect the mDNA stage, it must be radioactive and it must be myosin specific. We use an enzyme, reverse transcriptase, to make a copy of the myosin mDNA. This is labeled radioactively to be able to detect the stage. There are six (6) stages are differentiation. All the stages are separated. We isolate DNA from each stage and then run on the gel. We blot those RNA onto a membrane filter and use a probe to find the mRNA. (diagram shown to show stages) Myosin is not found in the early stages, myosin is found right after fusion and up until the muscle twitching stage at the end. This shows the myosin gene has been turned on after fusion. This proves the muscle specific proteins are activated after fusion. In situ hybridization: This experiment identifies the cell type in which the myosin first appears. We hybridize the muscle cells that are growing in culture and react them with a cDNA probe. Holes are put into cells and cell is hybridized. We can then see what cells have been bound to the cDNA probe. The myosin gene is not turned on until after fusion and before fiber formation. Slide 10, How do we examine changes in myosin protein profile? When do these proteins first appear? We can perform Western Blotting and/or Immunocytochemistry, which identifies the stage where they first appear. Western Blot (WB): We need the myosin specific antibody which will detect the myosin protein. We make this antibody by isolating protein from the rat and injecting it into another animal (rabbit), the rabbit reacts by making an antibody and this can be isolated and harvested from the rabbit.
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