Review for the third hour exam, part one 1) What are three embryonic organs whose medio-lateral, anterior-posterior and dorso-ventral axes become irreversibly decided during early development? Hint: Limb buds, inner ears, retinas 2) Are all three axes decided simultaneously, or one at a time, or sometimes one at a time, but other times simultaneously? 3) Why do limbs branch into three when a limb bud is grafted backwards? 4) How could you produce a 6 legged salamander (or chicken)? (Perhaps as extras in a John Carter of Mars movie?) 5) Draw a sketch of the Apical Ectodermal Ridge in a bird embryo. Contrast the AER structure in embryos of birds, mammals, frogs, salamanders, and fish. 6) Can you figure out any logical reason why the same category of protein (Fibroblast Growth Factor) induces third limbs and also causes the Medio-Lateral axis of limb buds? (I don't know myself why this should be true.) Hint: Substituting for the AER in maintaining limb proximo-distal development is kind of like inducing an additional limb to form. . *7) Based on your knowledge of the shape of cross sections through the tips of developing vertebrate legs (including the shape of the apical ectodermal ridge), and also based on what you know about relations between surface curvatures, tensions, and pressures, and the abilities of curvatures and tensions to vary as a function of direction, suggest combinations of changes and differences in tensions and curvatures of limb bud surfaces could explain their shapes. Suggest experiments that could test your hypotheses. 8) If the surfaces of limb buds contract with equal strength in all directions at all locations, and if their inside pressure is equally strong everywhere, then those combinations of mechanical properties would cause limb buds to become what shape? Hint: hemispherical 9) Based on the rule that the tensions in the surfaces of cylindrical tubes are exactly twice as strong in the circumferential direction as compared with the tension in the longitudinal direction, by means of what changes do limb buds change from being hemispheres to becoming round-ended cylinders? For the same reasons, how do hemispherical aggregates of Dictyostelium amoebae change into long cylindrical "slugs" Hint: By doubling their surface tension in the circumferential direction relative to surface tension along the proximo-distal axis (the same thing as medio-lateral axis) 10) What combinations of symmetry do each of the following have? Limb buds before they develop an apical ectodermal ridge Apical ectodermal ridges Hint: a long narrow ridge less symmetrical than either a flat place or a hemisphere) . Would you consider that formation of an apical ectodermal ridge is an example of "symmetry breaking"? 11) Think about some alternative explanations for Apical Ectodermal Ridges: Recapitulation of the evolution of legs from fins A side-effect of directional changes in tension needed for leg elongation A means of breaking symmetry of limb structures A boundary between dorsal and ventral sides of limb buds Please try to invent some others... 12) When a salamander limb regenerates, the muscles near the cut surface "dedifferentiate" (become undistinguishable from cells that had been skeletal cells), and then grow and divide until the cell mass is nearly as big as the amount of tissue removed. Then muscle cells re-differentiate (only) into muscle cells, and skeletal cells redifferentiate (mostly) into skeletal cells. Based on these facts, What can you conclude about the mechanism of pattern formation in limb buds? Is it by rearrangement of cells according to cell type? Hint: yes. Or is it by re-differentiation of cells according to position? > What can you conclude about the usefulness or need for undifferentiated stem cells? 13) Compare these alternatives to what H. V. Wilson hypothesized about the reformation of functional anatomy by dissociated sponge cells. Hint: Wilson assumed that sponges re-formed by re-differentiation of the equivalent of stem cells, or at least by switching from one cell type to another; he hated the idea that being differentiation causes cells to move actively to their correct relative locations, although rearrangement is really what happens, and is also what Wilson is credited with discovering. 14) By radioactive labeling just one cell type at a time (e.g. just the muscle cells or just the chondrocytes (= cartilage cells), researchers discovered that cells don't switch from one cell type to another during regeneration. (New muscles are made of cells that had been muscle cells in the cut off stump, etc.) Assuming this information is true, then argue pro or con whether regeneration of limbs should be considered logically equivalent to the sorting out of dissociated cells. 15) Argue pro or con: If the muscles of a regenerated leg consist entirely of cells that were muscles in the stump, and if all the skeletal cells in the stump become skeletal cells in the regenerated leg, that means that regeneration results from rearrangement of differentiated cells, instead of what most people assume (spatial control of undifferentiated stem cells). 16) What would be some medical uses of a method that could cause cells of one differentiated cell type to convert to cells of a different cell type? 17) Describe the sequence of events that occur when a newt or other salamander regenerates one of its legs. 18) What is a blastema? Where do blastemas develop? Suppose you put a blastema into tissue culture. What might happen? 19) When salamanders regenerate the skeleton and musculature of one of their legs, do any of the previous chondrocytes redifferentiate as muscle cells? Hint: No? What is some experimental evidence that would support or contradict this interpretation? 20) Likewise, do any of the previous muscle cells redifferentiate as skeletal cells? And what evidence would be needed to prove or disprove this? *21) Discuss why, or why not, you would or would not have expected these results, drawing on several particular facts and principles that you have learned in other parts of the course. *22) What are some facts that would have led you to expect these results? *23) In terms of each of the following hypothetical phenomena, please suggest possible reasons why salamanders can regenerate legs but mammals, birds, frogs and reptiles cannot regenerate legs. And for each of these hypothetical explanations, please propose at least one experiment that would be capable of either confirming or disproving the theory. a) Maybe (in mammals, birds and reptiles and frogs), all their myoblasts differentiate into myotubes (=skeletal muscle cells), leaving no undifferentiated myoblasts, but even in adult salamanders enough undifferentiated myoblasts remain (enough to provide the muscles for the regenerating legs)? b) Maybe only salamander muscles can dedifferentiate and separate back into undifferentiated muscle cells? c) Maybe only salamander leg cells continue to be able to crawl and exert traction, sufficiently to rearrange leg cells into their correct anatomical patterns? . d) Maybe only salamander tissues continue to be sensitive to "Positional Information"? e) Maybe only salamander tissues continue to produce "Positional Information"? f) Maybe only salamander cells can switch from one cell type to another? g) Maybe only salamanders do not need an apical ectodermal ridge in order to form a leg, and (also maybe) no vertebrate (except tadpoles) can re-form a new apical ectodermal ridge? Hint: Maybe none of them can regenerate new AERs, but salamanders don't need an AER. (And, yes, I know that regenerating salamanders do form a thickened cap, but not an AER.> 24) Compare or contrast the three kinds of explanations that scientists have advocated as for sorting out of dissociated cells. (including Malcolm Steinberg's theromodynamic explanation.) [hint: are there logical similarities or equivalences in the theories, and in the kinds of evidence that are needed to prove which explanation is correct?] 25) If you dissociate a neural tube into its individual cells, and then randomly mix these with dissociated cells from the "somatic ectoderm" (i.e. the cells that will form the outer layer of the skin), then the neural tube cells will sort out to the internal position. Argue pro and/or con whether this means that the cause of cell sorting must have the same underlying mechanism as the cause of normal neurulation. Remember that embryos of mammals, salamanders, and frogs form their neural tubes by folding of a sheet of ectodermal cells, and sealing the edges together, leaving a fluid-filled space inside, called the neurocoel. In contrast, embryos of teleost fish form their neural tube from a solid rod of cells, which somehow hollows itself out down the middle (that is, instead of folding and sealing the edges). Argue pro and/or con whether this means that teleost neurulation has a different causal mechanism than the neurulation of mammal embryos, frog embryos and salamander embryos. 23) Briefly describe and/or discuss whether your opinion or conclusions on the questions above are changed by each of the following facts: a) When sorting-out occurs between neural tube cells and somatic ectoderm cells (in any vertebrate), the mass of aggregated neural tube cells hollows out to form a neurocoel-like cavity. b) The anterior 80% of a bird embryo's neural tube forms by active folding and sealing of the edges, just as occurs in the whole neural tube of mammal and amphibian embryos; but the rear-most 20% of a bird embryo's neural tube forms by hollowing out of what starts out as a solid rod of cells. c) If a solid ball of aggregated neural tube cells is put in tissue culture media, next to and in contact with a solid ball of aggregated somatic ectodermal cells, then the latter will extend around the outer surface of the mass of cells derived from the neural tube, until it forms a continuous surface layer. (i.e. the same arrangement as gets produced when these two kinds of cells sort out). 24) Argue pro or con: Cells of the eye can be observed to sort out from cells of limb buds (with which they normally never come in contact), therefore we can conclude that the mechanism by which cells sort out cannot be important in normal embryonic development. (There is nothing to learn from cell sorting about the causes of normal embryonic development?) 25) According to material posted on the web page, which particular scientist reached this conclusion while stopped at a traffic light? 26) How would (and did) Malcolm Steinberg interpret the ability of dissociated and randomly-mixed cell types to sort out from each other, regardless of whether any given pair of cell types ever normally come in contact in normal development? 27) Because normal animal development never consists of formation of 5 or 6 concentric spheres of differentiated cells, layered around each other, therefore would you argue that the causal mechanism of cell sorting can't be the same as (or over-lap with?) the causal mechanism of normal development? 28) Because no kind of animal develops from an embryo consisting of a random mixture of all kinds of differentiated cell types, which then sort out so as to create anatomical structures, would you say that therefore the mechanism of cell sorting can't be the same as the mechanisms of normal embryonic development? 29) Because mixtures of differentiated cells often rearrange by means of several different sequences of geometrical intermediates, on their way to form the same eventual anatomical shapes, that tells us what? More than one of these conclusions might be true.(Don't panic: if these questions are on an exam, they will be graded leniently). 1) That different causes can form the same end result? 2) That the same causes can produce different intermediates? 3) That cell sorting is caused by lack of counter-balance between opposed forces? 4) That these forces eventually become balanced? 5) That there are many arrangements in which opposed forces are imbalanced? 6) That (in such a case) there was just one balance in which forces are stably counter-balanced? 7) That the forces producing the rearrangement must all be thermodynamically reversible? 8) That only reversible forces can produce the same end result from different beginnings? 9) That different kinds of cell-cell adhesion proteins must cause sorting out. 10) That different total amounts of adhesion proteins (regardless of kind) must cause sorting out. Which are true? Which are true and can be logically deduced from the observed ability of cells to reach the same eventual results by two or more different sequences of intermediates? Which are not true? Which are not true, but believed by many scientists? Which could or would be true, if some particular additional fact were observed or otherwise known? 30) What are at least five normal functions of programmed cell death (apoptosis)? 32) The protein bcl-2 has what effect on programmed cell death? 33) How was bcl-2 discovered, and how is this method of discovery related to the name? 34) What is the distinction between necrosis versus apoptosis? 35) What are caspases? How are they related to programmed cell death? 36) Suppose that you could invent a treatment that would activate caspase in any cells that had an abnormality that only occurs in cancerous cells?