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Lecture 7

LECTURE 7: Muscle and Liver Development.doc

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Biological Sciences
Ian Brown

BIOC19: LECTURE 7 Slide 2: • Skeletal muscles a.k.a voluntary muscles • Myofilaments are composed of muscle specific proteins: actin and myosin. Slide 3: • Pink diagram shows the development of the neural tube. The ectoderm folds-in, forms the neural groove, and then the neural tube pinches out. This diagram shows the cross-section of the neural tube. • Black diagram shows the top view of the neural tube. The early neural groove is not zipped and the neural tube on its right is zipped up or closing neural tube in its late phase. Its starts to zip at the brain and then to the posterior end of the embryo. Neural tube zippers up before the development of muscles. • The diagram on the lower left also shows a cross-section of a neural tube. In that diagram, neural tube is pinched. Right under the neural tube is notochord and the red part is mesoderm, which eventually develops into different regions. The red mesoderm cells proliferate and surround the embryo. There is also endoderm in the middle and epidermis on the outside. Slide 4: • Diagram with labels show locations or action sites of mesoderm regions. Note: the missing label is "Axial Mesoderm" • Somites form into muscles • The 3 main regions are found on both sides of the central neural tube (refer to the diagram) Slide 5: • Upper right diagram shows a microscopic picture. It shows the neural tube in the middle and sausage-shaped paraxial mesoderm on both its side • Paraxial mesoderm starts diving into somites (the bumps in the upper right picture), starting from head and moving down to the tail sequentially • Different species of animals has different number of somites • All vertebrates have division of paraxial mesoderm • By counting the number of somites, you can tell what stage of development a particular animal is at because as development progresses, the number of somites increase Slide 6: • In the first diagram, you see the pre-segmented stage. Pre-segmentation stage is before the division of paraxial mesoderm into somites when the neural plate is still open, the neural groove is still there, notochord is present, and there is unsegmented mesoderm (particularly paraxial mesoderm) • Then, in the second diagram, you can see that the neural tube closes and pinches off. The unsegmented mesoderm starts cutting off individual somites. As you can ssee, somite is a mass of cells • Then, in the third diagram, you can see that the somite differentiates into two components. The part closer to notochord and the bottom of the neural tube differentiates into sclerotome. The part of somite that is more closer to the surface or more dorsal differentiates into dermamyotome. • Dermamyotome subdivides into 2 further regions: dermatome and myotome. Dermatome is closest to ectoderm and it forms the dermis layer of skin or the deep layer of skin. The more internal, larger mass of dermamyotome subdivides into myotome which forms the skeletal muscles. Slide 7: • This experiment proves that the cells in the somites form muscles of the limb/chicken wing • Note: The blue thick double line on the far right of the diagram is Neural Tube • Diagram on the far right shows the limb bud of the chicken that forms the wing • Surgically remove chick somites and replace them with quail somites which have a different pigmentation than chick somites. Then look at what develops. Result: a wing develops and we can see that the wing's cells have striated muscle which all have the pigmentation of quail somite. So, skeletal muscles of the limb originate from somites because as shown in the experiment, limbs of the chicken were derived from cells that originated from the quail somites Slide 8: • Myogenesis is the development of skeletal muscle tissue. It can be divided into 3 stages • Commitment/Determination is when pluripotent commit to the myogenic pathway because otherwise pluripotent cells can differentiate into many other things • Differentiation steps involves turning on genes encoding skeletal muscle protein genes • Skeletal muscles are the only type of cellular body that can contract. Their ability to contract is controlled and triggered by nerve action. That's why nerves must grow into the developed muscle cells to form a neuro-muscular junction. The firing of the motor neuron controls, through the neuro-muscular junction, when the muscle is going to contract. Slide 9: • #1 sentence refers to the diagram on the far left. In fact, each sentence refers to a diagram on the top • Note: On the diagram that is on the far left, label the blue line 'Neural Tube' and the red circle 'notochord' • Shh is an inducing factor, given off by neural tube and notochord, which triggers step# 2 • In step #2, Shh transforms myotome cells to myoblasts, triggering commitment to the myogenic pathway • In step #3, myoblasts undergo rapid cell division and when they reach the right number, they begin to align and undergo cell fusion so lots of myoblasts end up in one cell, giving the cells its contractile ability and multi-nucleated characteristic • Muscle specific proteins include actin and myosin • Final step is when muscle contracts in a organized manner • Bottom picture is of muscle fiber cells. See how the many nuclei get pushed to the edge. • Slide 10: • There are very distinct series of steps in muscle differentiation, which can be recognized at the morphological with a microscope. They are also recognizable the individual steps at molecular level because of muscle specific proteins actin and myosin that form the myofilaments (the contractile element). At the molecular level, you can see when genes of actin and myosin are turned on and when those genes make the actin and myosin proteins. • Certain features of muscle cell differentiation are self-stimulating and we can look at the them in tissue culture without having to add external inducer AS LONG AS WE START WITH MYOBLASTS. In the last slide, we mentioned that myotome cells are pluripotent and Shh transforms them into myoblasts and triggers commitment to the myogenic pathway. That is why we would not have to add an external factor/inducer if we start with those committed myoblasts because we know that they are already committed/restricted to the myogenic pathway. Slide 11: • In this experiment, we're going to look at the differentiation of myoblasts in mature muscle fiber cells. • We start with embryonic thigh muscle that was isolated at a step when myoblasts were present. • So, we take a piece of the embryonic thigh muscle, cut it into tiny pieces, and then treat with an enzyme called trypsin. Trypsin digests the cell adhesion proteins that are holding the thigh muscle cells together and thus, the thigh muscle tissue is dissociated into individual myoblast cells. • Then you remove trypsin through centrifugation and you are left with single celled myoblasts. • You put these myoblasts on a petri dish (along with culture medium) and they undergo muscle cell differentiation without any external inducer. • In vitro mean tissue culture Slide 12: • What happens, step by step, when you put the myoblasts on that petri dish? (refer to experiment on the previous slide) • With such a tissue culture, you can recognize the different steps of differentiation at the molecular and morphological level • 2) Cell division stops when the cells reach a certain number/density of myoblast cells and through regulation, they stop dividing • 4) Cell fusion signals the change in the pattern of gene expression to turn on genes encoding actin and myosin • 5) Increase in muscle-specific proteins that organize themselves in myofilaments • 6) Twitching is spontaneous and not in an organized way because motor neurons have not yet grown in the muscles and neuro-muscular junction has not yet formed Slide 13: • Multinucleated cells form in 6-12 hours which is too little time for many many rounds of DNA replication for nuclear division • No mitotic figures or condensed chromosomes were observed under the microscope which we would expect to find during nuclear division • Multinucleated cells formed even if you put in DNA synthesis inhibitors which prevent nuclear division Slide 14: • Can different types of muscle cells fuse? No. Myoblasts of different organs or origins do not fuse together to make multinucleated cells • Note: the slide should say 'Thigh muscle myoblasts + heart muscle myoblasts = no cell fusion' Slide 15: • In this experiment, we're trying to see whether myoblasts, that are from the same tissue but different species, fuse. • Rat thigh muscle myoblasts labelled with radioactive triated thymidine + Rabbit thigh mus
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