Why branch into three when a limb bud is grafted backwards?
Hint: response of anterior tissue to being grafted very close to posterior tissue
How could you produce a 6 legged salamander (or chicken)? (Perhaps as extras in a John Carter of Mars movie?)
implant sources of fibroblast growth factor.
Draw a sketch of the Apical Ectodermal Ridge in a bird embryo in cross-section. Contrast the AER structure in embryos of birds, mammals, frogs, salamanders, and fish.
*Can you deduce what might be special about the tension in the surface membrane at the AER, relative to other parts of the skin? (There is more than one possibility.)
Can you invent experiments capable of proving and/or disproving which possibility is true?
What happens to limb development if you surgically remove the apical ectodermal ridge at different stages of development?
What happens if you graft a second apical ectodermal ridge to a developing limb bud?
What is unusual about the apical ectodermal ridges in salamander limb buds?
They don't exist
What else is unique about salamander legs?
They can regenerate
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.
What do you conclude about the mechanism of pattern formation?
Is it by rearrangement of cells according to cell type?
Or is it by re-differentiation of cells according to position?
Compare these alternatives to what H. V. Wilson hypothesized about the reformation of functional anatomy by dissociated sponge cells.
Hint: Wilson preferred to believe that sponges re-formed by 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 what Wilson is credited with discovering.
Sketch a mammal or bird limb bud, at 3 or 4 stages of embryonic development.
How can you cause vertebrates to form 3 legs along each side, instead of two?
How can you cause legs to branch into three hands, wings or feet? How can you cause them to branch into four distal ends?
Hint: by grafting what? to what location?
Who discovered both these phenomena?
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 am not sure 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.
What about the paracrine protein Wnt? What axis does it control in developing limb buds?
What properties of the neural retina does this axis correspond to?
Geometry of innervation of the brain by axons from retinal ganglion cells.
*Imagine an experiment in which one "hand" of a salamander is amputated, and then grafted to the elbow of another salamander's "arm", or if the hand were grafted to the shoulder, or anywhere along the length of the "arm". (This was done by Nardi and Stocum, at the University of Illinois. It was discussed in the lecture but their names aren't mentioned in the notes on the web). What happens is that the grafted hand moves very slowly, day by day, out the length of the "arm", toward the normal hand, until it is connected at the wrist, at the location next to the normal hand. The reverse experiment has also been done, with analogous results.
Invent a method for using fibroblast growth factor to change the result of the Nardi and Stocum experiment.
How are Nardi and Stocum's observations related to the question of rearrangement versus redifferentiation?
Hint: Rearrangement of cells and matrix requires mechanical forces; migration of limb explants along the leg proves the occurrence of physical forces acting at the right time in the right places.
If you sliced a salamander limb bud like a long bologna sausage, and cultured each of the slices onto a thin sheet of rubber (to detect amounts and directions of contractility), what differences would you expect, based on the result of the Nardi and Stocum experiment?
Hint: Hand cells should contract more strongly than elbow cells, and much stronger than shoulder cells, according to the Brodland-Harris counter-theory
Could the same Nardi and Stocum effect be produced by gradients of strength of contractile strength of a developing limb bud?
If forces don't create shapes, what does?
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).
Argue pro or con: Will stem cells be able to regenerate complicated anatomical structures? (instead of just particular cell types, that either have a very simple geometry, like skin and intestine, or have no geometry, like blood)
Concentration gradients can be produced by diffusion (for example auxin, sonic hedgehog and retinoic acid). But how can embryos form gradients of substances like eph and ephrin surface proteins, or messenger RNAs of transcription factors?
Hint: remember that computer program I projected in class.
Compare and contrast the reversibility of differentiation in animal cells versus plant cells.
* 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?
Describe the sequence of events that occur when a newt or other salamander regenerates one of its legs.
What is a blastema?
When salamanders regenerate the skeleton and musculature of one of their legs, do any of the previous chondrocytes redifferentiate as muscle cells?
What is some of the experimental evidence for or against this fact?
Likewise, do any of the previous muscle cells redifferentiate as skeletal cells? And what evidence would be needed to prove or disprove this?
*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.
*What are some facts that would have led you to expect these results?
*Conversely, what facts would have led you to expect that dedifferentiated blastema cells would re-differentiate into whichever differentiated cell type is needed at each particular location?
In what ways is what happens in salamander limb regeneration like what happens when dissociated sponge cells sort out?
Important: Compare the limb bud development with development of the entire animal of sea urchins, frogs, and mammals.
Hint: What happens when limb buds, and embryos are split in two, or fused side by side?
Also, compare or contrast the development of anterior-posterior axes in limb buds as compared with whole embryos.
Imagine an experiment in which somebody takes an early snake embryo, soaks a plastic bead in fibroblast growth factor, and then surgically implants this bead into the somatic layer of the lateral plate mesoderm, and the snake then develops a leg, with fingers and claws, at the location where the bead was implanted! What might this result tell you about the molecular changes in evolution by which snakes stopped developing legs? (Specifically, as opposed to what other molecular changes that could have produced this same result).
Hint: Remember Koller and Fisher's experiment involving bird teeth?
Suppose that at the one cell stage of an embryo, two chromosomes happen to break at locations between promoter regions of certain genes and the parts of those genes that code for the protein, and then each chromosome fragment rejoins incorrectly with the DNA of the other chromosome. If the genes are normally transcribed only in differentiated cells (and NOT in the same cell type), then what will happen as a result of this genetic translocation?
*Imagine that you are one of Driesh's "entelechies" whose job it is to adjust the sizes of the parts of embryos that have been separated into halves, or quarters, or fused into double-sized embryos. Could you accomplish this goal by adjusting the diffusion rates of "morphogen" chemicals? Hint: yes; for example in an embryo cut in two, each of its entelechies could make morphogen "B" diffuse twice as fast as in a normal embryo, so that the normal number of somites (or any periodic structures) would form in half the distance, with each one being half as big as normal, and made of half the amount it tissue.
*Continuing this fantasy of being an entelechy, if embryos control organ location by "positional information" (in Wolpert's sense), then how would you need to change the gradients of morphogens in separated embryo halves, or quarters?
*If the concentration gradient of a morphogen formed because messenger RNA for the morphogen was concentrated at one end of the embryo, then how could cutting the embryo into two result in formation of two concentration gradients, each twice as steep as normal? Or can it?
Hint: Frankly, I don't see how.
*Remembering how the mammal body develops from the primitive streak in the inner cell mass, would you expect a morphogen gradient to have its maximum where the primitive node forms? Or would its minimum form there? Hint: in principle, it could work either way.
Suppose you were to dissect two early chicken embryos out of their eggs, into tissue culture dishes, and lay one embryo right on top of the other: then make a time-lapse video of somite segmentation in both embryos. You might need to focus up and down frequently, so as to see both the upper and the lower embryo. Alternatively some very long working distance microscopy might be possible - or confocal microsopy? The goal is to find out whether the embryos somehow influence each other's times of somite separation. For example, these times might tend to synchronize! Would you interpret synchronization as evidence supporting the clock and wave-front hypothesis? Or would synchronization be evidence in favor of control by a reaction-diffusion system? Or would synchronization be predicted by both those theories?
Would it matter whether both embryos were lined up, with heads in the same direction? What if you laid them down at right angles, one perpendicular to the other? What if you laid them down in opposite directions, with the head end of one directly above the tail end of the other embryo? What if you laid them down with the head of one over the neck of the other; or with the head over the middle of the other?
What if you had different mutant strains, one of which formed more somites than the other? Or one of which formed somites faster than the other? ...and you plop one isolated embryo directly on top of the other. The goal is to create a situation in which the different alternative theories make sharply different predictions.
**Think about stripe color patterns in certain fish: Argue in favor or against as evidence that these stripes are caused by the sort of mechanism invented by Turing.i.e. that Turing mechanisms can't explain most anatomical patterns because Turing can't easily explain what Driesch discovered; but the stripes in the skins of this kind of fish stay the same size as the fish grow, and more stripes form.
What is the biochemical mechanism of programmed cell death?
List as many specific examples as you can of apoptosis.
How was genetic research on the nematode C. elegans essential for the discovery of the biochemical cause of apoptosis?
Why was it important that C. elegans coincidentally happens to have only one gene for a caspase, rather than 2 or more?
What are at least two connections between cancer and apoptosis?
TO BE CONTINUED