Embryology Biology 441 Spring 2010 Albert HarrisLecture Notes for March 24
Bones get stronger in response to exercise, and related phenomenaA familiar fact. (
Thickening of skin as a local response to friction is another familiar phenomenon.Again, however, what is the molecular mechanism? Is stress, or strain, or damage, detected by skin cells?What difference makes the palms of your hands and soles of your feet so much more responsive to this mechanism?
Fewer people realize that similar strengthening also occurs in cardiac muscle, in bone, and perhaps in artery walls(and maybe also in cartilage and other tissues and organs). Effects of such stimulation by function can be either good or bad.For example, strengthening of smooth muscles in the walls of arteries in response to increased blood pressure can produce a vicious cycle Cardiac muscles also increase thickness and strength in proportion to "load", in the sense of how much force they are required to exert. For example, before birth the wall of the left ventricle is no thicker than the wall of the right ventricle. The reason is believed to be that both ventricles were pumping against the same resistance, and the same blood pressure, because of (what you have already learned) about the foramen ovale and the ductus arteriosus. After birth, the strength and thickness of the muscular wall of left ventricle increases gradually until it is about 4 times stronger (and thicker) than the muscular wall of the right ventricle. This matches the approximately 4-fold higher blood pressure of the systemic circulation, as compared with the lower pressure of the pulmonary (lung) circulation. One might have expected that, perhaps, the right ventricle would get weaker, instead of the left ventricle getting stronger? My best guess would be that it's because the whole heart, and the whole body, are growing so much after birth that neither side can be expected to become weaker. Incidentally, the curvature of the outer wall of the right ventricle is much greater than the curvature of the left ventricle. Until I can include a photograph or at least a drawing of this (very dramatic!) difference in curvatures, the best I can do is a schematic use of two letters of the alphabet, upper-case C to indicate the right ventricle, and lower case o to indicate the left ventricle.
CoImagine if you had two rubber tubes, glued strongly together along one side, one tube containing low pressure, and with a thin wall, and the other tube with a thicker wall and higher pressure, this is the geometrical arrangement that would be created by the differences in pressure and tensions in the rubber tubes. The high tension tube would pull the wall of the low tension tube around itself, and the high pressure tube would also bulge sideways into the lumen of the low tension tube. Some of the most important discoveries about artery development in recent years have been made by Dr. Rong Li, now doing research in California, who got her PhD in this department working with Prof. Bauch, and took this course.
BoneBone also responds to stress by becoming stronger: this is not so widely known, as muscle strengthening!Researchers have reported measurements of calcium phosphate amount in the arms of former professional-level tennis players, and found 30% more calcium phosphate in the bones of the right arms of right-handed tennis players. The humerus, radius and ulna all have thicker and more dense walls. The trabeculae inside these bones also become thicker and more numerous. Another researcher tested the effect of surgical removal of either the radius or the ulna from sheep's legs, followed by letting the sheep recover and walk around for weeks or months, and then sacrificing the sheep and making histological sections of whichever lower leg bone had NOT been removed. The idea is that the one remaining bone will have carried the whole load, about double the stress that it normally carries. As expected, these heavily-loaded bones rapidly enlarged in size and strength. A surprising result was that spongy bone rapidly formed on the outer surfaces of these doubly-loaded radiuses and ulni. This was the first stage of the response, and was followed by formation of dense layers of bone surrounding the new spongy bone. Within a few months, the remaining doubly-loaded bone had increased in diameter and strength enough to make it is strong as the combined radius and ulna. Conversely, bones get weaker if they aren't subjected to loads. Examples are when people are immobilized for long periods, when broken bones are set in casts, and when astronauts are in space. NASA likes to call weightlessness "microgravity", as though the latter were the technical term. Actually, it's just wrong. Such words can serve an invaluable function as detectors of well-meaning ignorance. Weightlessness somehow causes bones (and muscles!) to get weaker, within days and weeks. The mechanism isn't known, but has traditionally been assumed to be the reverse of the strengthening of bones subjected to stronger-than-usual forces. I suspect this is a mistake, because the weakening is much faster than the strengthening, is much more extreme, and can only be slightly overcome by exercise. NASA is a lot better on rockets than biology. Major discoveries are waiting to be made on these subjects, and NASA offers very generous grant funds if you can think of even half-way good experiments. At their expense, I once attended a week-long conference on "microgravity" in New London, New Hampshire, where I met some excellent scientists and found a wonderful used book store near Lake Sunape. For more than 30 years, but without adequate evidence, the mechanism by which bone cells detect forces has been claimed to be that electrical voltages are produced by the phenomenon of piezoelectricity, and that bone cells respond to these voltages by producing more bone wherever the voltages are largest, and maybe aligning some of the bone produced parallel to these voltages. Machines were built, sold and used in hospitals that applied small voltages through the skin of patients with broken bones. Nurses and MDs have told me that they didn't think these machines have much effect, if any. Whether this approach becomes misused by professional athletes may be the surest criterion for whether externally-imposed voltages really can induce bone formation and strengthening! Many different crystalline substances, including quartz and titanate salts generate temporary voltages when either squeezed, stretched, or bent. If you know what phonographs are, the cheaper of the two ways to convert the grooves in records into electrical voltages was by twisting barium titanate crystals. (The higher-fidelity method used magnets and electric coils; in case you like to know how things work) CDs work by destructive interference of laser light reflected at dents adjacent to flat places. CDs are cheaper to make, but don't last as long as vinyl records; in both cases the reverse of what everyone assumes. Sonar "pingers" and sonicators work by applying a voltage to quartz or other piezoelectric crystals, because piezoelectricity works in reverse: voltage produces shape change, and vice versa. Bone is only slightly piezoelectric, and (I have read, but tend to doubt) it's the collagen part of the bone that produces the voltage. Calcium phosphate, by itself, is not supposed to be piezoelectric. Most people tend to think a voltage produced by bone must be serving some function, tending toward credulity about theories of bone growth and strengthening being stimulated by voltages. Few biologists realize that compression or stretching of cartilage produces much larger voltages (than bone), as much as hundreds of volts. That it because squeezing water out of a cartilage causes positive counter-ions to be carried along with the water, thereby creating a temporary negative voltage inside the cartilage, and a positive voltage outside. The same will occur with any material that has stationary ions that happen to be either predominantly anions or predominantly cations. Therefore, collagen gels and bone might produce voltage by electro-osmosis, when compressed or stretched. In other words, the effects that people have been reporting might not be piezoelectric at all. Another aspect of these phenomena that most people don't understand is that the voltages are only temporary. They drain away in a fraction of a second. Thus any measured voltage in proportional to the rate of distortion, not the absolute amount. Furthermore, you get a reverse voltage when you relax the distortion. Thus, to the extent that bone can respond differently to compression than to tension, or respond to steady forces rather than rate of change of force, the signals can't be results of either piezoelectricity or electro-osmosis. A steady force won't produce any voltage, by either mechanism; and the reduction of compression produces a voltage indistinguishable from an increase in tension; nor will an increase in compression produce a different electric field than release from being stretched. Likewise, those machines for applying voltages to broken bones ought to have been applying AC rather than DC, but they may not have worked at all. The best scientists have tended to stay away from this whole subject, unfortunately for patients with broken bones and osteoporosis. Try to find either the word electro-osmosis or the word piezoelectricity in the index of books on embryology or developmental biology. Both words are defined incorrectly in the dictionary of this word processor. The definition of piezoelectricity is fairly close, except that what is produced is a voltage, rather than a current. The other word's definition is wildly misleading; membranes aren't involved. The whole point is that electric fields substitute for membranes. Big medical advances could be made with just a little more understanding.
An interesting special case: kidneys.If one of the two kidneys is surgically removed from a human or other vertebrate (as for a transplant), then it has been observed that the remaining single kidney rapidly doubles in size. You don't get any more tubules, however. This enlargement ("hypertrophy") was assumed to be a response to the increased load, in the sense of the need to filter-out all the waste molecules, rather than filtering-out only half.In graduate school I was taught about an elegant experiment that disproves that interpretation (but unfortunately failed to learn who did the experiment, or where and when it was published). The experiment consisted of surgically cutting one of a rat's (?) two urine ducts ("ureters"), so that urine from that kidney flowed into the coelomic cavity, and eventually back into the blood. Both kidneys were then forced to work twice as hard (because the kidney with the cut ureter was accomplishing nothing). The expected result would have been that both kidneys would double in size; but in fact, neither kidney enlarged! That result would have fit a then-popular concept of self-inhibition of growth, somewhat analogous to auxin's inhibition of branching in plant shoots. One hypothesis was that each kidney secretes an inhibitor of kidney growth, that the amount secreted is proportional to the number (and total size?) of kidneys, and that this inhibitor increases in concentration in body fluids until it's concentration is just barely sufficient to block further enlargement of kidney tissue. Removal of one of the kidneys would (according to the theory) result in less inhibitor being produced, so that the concentration is decreased, which allows the remaining kidney to grow larger, with the amount of this growth then being limited by its own production of more inhibitor. Among the foggy areas of this theory are proportionalities between concentrations of inhibitor and rates of secretion of inhibitor. Does the stuff slowly degrade, or is it excreted by the kidney? Do those questions need to be asked, as a necessary stage of designing experiments to test the theory? Or can we wait until the theory has been proven true, before asking too much about such details? Also, is isolation and purification of the hypothetical inhibitor a necessary first (or early stage) in proving whether the theory is true or false? What do you think? How would you find the inhibitor, if it exists? By what methods (bioassays? Genetic screens? Gene knock-outs? Genome sequencing? Stem cell research? Something else!) would you try to test this theory of "de-inhibition" of kidney size? I should mention that there is another theory, which is almost a mirror image of the one described above. This other theory (or other category of theories) is that some particular chemical substance either stimulates kidney growth, or is somehow necessary for kidney growth, and this stimulator is produced by some other part of the body, and is destroyed or inactivated by the kidneys - destroyed at rates or in amounts proportional to the number and size of kidneys in the body. Thus, removal of one kidney would reduce the rate or removal of this stimulatory substance, so that the stimulatory substance would increase in concentration, therefore stimulating the enlargement of the remaining kidney, until it reached a big enough size to absorb, or destroy or inactivate so much of the stimulator that kidney enlargement would be halted at about the same total volume as the sum of the original two kidneys. Questions for class discussion: 1) For every hypothesis based on stimulatory or inhibitory chemicals or inhibitors, is there always a mirror image theory that could potentially explain the same facts? 2) Given such a pair of mirror-image alternative theories, what sort of initial experiment should be able to distinguish which of the two mirror images is closer to the true explanation for the phenomenon? 3) Which would be easier to identify conclusively (for example, using a bioassay method) a stimulatory chemical or an inhibitory chemical? 4) What about the specificity of the effects of chemicals being tested? Might lots of unrelated chemicals just happen to inhibit the enlargement of kidneys or other specific organs, whereas only a few chemicals (whether chosen at random, or purified from ground-up tissues) would stimulate growth of kidneys. Is this likely to be even more true for stimulation only of growth of kidneys, with the isolated or chosen chemical not stimulating growth of any other organ or tissue. 5) Suppose that a mutation was found in mice (or a rare genetic syndrome was discovered in certain related people) which produced abnormally large kidneys.
(i.e. What would you hypothesize? How would you relate this observation to past theories?)
2) What if this protein (coded for by the gene that is mutated in the mice and people with the abnormally-large kidneys)
were purified and then its survival was measured while placed in tissue cultures of kidney, tissue cultures of liver, etc. 3) What could you learn from measuring the relative amounts of this protein produced in kidney tissue versus liver, versus other tissues? 4) What could you hope to learn by using in situ hybridization to compare rates of transcription of this protein in sections through animal tissues 5) Would it be worthwhile to compare the distributions and amounts of messenger RNA for this gene in sections of embryos as compared with sections of adults? 6) What about comparing transcripts (m-RNA) of this gene in mutant mice versus wild-type mice? What could you learn?
7) Suppose that the mutation (that causes the big kidneys) is dominant, in the sense that mice (or people) who are heterozygous for the mutation would have kidneys just as big as those that are homozygous for the mutation?
8) Base sequences of genes usually can tell you the general function of the protein that the gene codes for, such as whether it is a transcription factor (serving to control expression of other genes), a kinase, or other enzyme, a structural protein, a cell adhesion protein, a cell-cell signal molecule (a growth factor or cytokine), or a receptor protein for a signal molecule. Or could it be any of this list of categories of proteins? In which case, outline a theory for how kidney size could be regulated by each of these categories of proteins.
A little about the skin-thickening phenomenon:In the 1970s, an English researcher name Bullough developed a bioassay in which adhesive tape was used to pull of the outer layers of skin of mice, and increased rates of division is skin cells was quantified by incorporation of radioactive DNA precursors. He favored the removal-of-a-growth-inhibitor category of hypothesis. As is usual, he invented a classical-sounding name for the hypothetical chemical inhibitor: "chalone". Don't laugh too hard, because the names serotonin and auxin and were invented long before these chemical substances were actually identified by means of bioassays. Serotonin is the basis of a multi-billion dollar drug industry and auxin is the basis of a multi-billion dollar agricultural industry. The hope was to isolate and identify some particular chemical that regulates skin thickness, by being secreted by skin cells and inhibiting thickening more than the normal amount. Unfortunately, negative bioassays have a tendency to fail, because many chemicals may inhibit growth without that being their function. There also wasn't much prospect of a multi-billion dollar industry.
Mammals, including humans, are very good at regeneration oflarge parts (2/3) of their livers!Nobody yet knows how this is controlled, either. Even for parts of your body that don't regenerate, it's a good question what mechanism halts their growth when they have reached normal size. Medical revolutions will eventually be accomplished.
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