Biology 441, Spring 2014


"The question of how patterns originate is the Gordian Knot of Developmental Biology."
Lewis Held, 1992

What are the forces that shape anatomical structures?

----> To say "Growth" is a major oversimplification. <---
In the history of embryology, growth was considered the explanation for many shape changes and cell rearrangements. For decades, the organization that is now named the "American Society for Developmental Biology" was instead named "The Growth Society"

None of these is "growth" in this simple sense.


What are the real embryological forces?

Water pressure ("hydrostatic pressure')
    Two good examples are inflation of the neural tube, especially the brain. and inflation of the eyeball and cornea.

    Evidence: If you puncture an early embryonic brain, water spurts out.
    Creating a permanent hole, through which water can leak out, prevents brain development.

    Eyeballs also stop enlargement if punctured, or if a small glass tube is inserted through the wall. Water flows freely out the tube. Constriction of surrounding tissue would normally seal off an opening.

    The amnion ("bag of waters") is also inflated by water being pumped into it.

    Synovial cavities in most joints are inflated with synovial fluid, to reduce friction. Fluid pressure of synovial fluid also supports some of the weight.

    By "pumping" I mean active transport of water through cells, analogous to what goes on in kidneys. I wish I knew more about the chemical processes by which ATP energy gets used to pump water. It isn't pinocytosis.

    Brain shape results from local differences in mechanical resistance to water pressure.

    For example, the lobes of the brain are caused by local weakness, that allows stretching of neural tube tissues, so that they balloon outward.

    The slightly greater curvature of the cornea of the eye results from slightly weaker mechanical tension, as compared to the rest of the eyeball.

    The heart changes its complicated shape at each stage of embryonic development, and also deserves to be included in the category of embryonic structure that are inflated by water pressure, and whose shapes are caused by counter-balances between outward pressure pushing against circumferential tension.


Osmotic Pressure: Swelling of individual cells

    This is also important in embryos.

    Not just red blood cells, but all cells of the body can easily be caused to shrink by dissolving inert sugars in their tissue culture media. Part of my own PhD thesis work used this fact to study mechanisms of "amoeboid locomotion ".

    Hypertonic solutions don't just shrink cells, but also dehydrate their cytoplasm and freeze all their surface movements and interior cytoplasmic flows. (Instantly, and reversibly!)

    This can be interpreted as evidence that osmotic pressure provides at least part of the force that pushes the front end of a cell forward.

    I think that's a true conclusion; but more research should be done. We really need some way of measuring how much forward force is exerted by crawling cells. Tissue cells definitely do a lot of pulling, which can be unexpectedly strong.

Electroosmotic Pressure: the driving force of cartilage expansion and elongation.

    Electroosmosis does not require or involve semi-permeable membranes. (amazing!)

    Ions are caused to be concentrated at high concentrations by electric attraction toward high concentrations of covalently-bound sulfates, attached to chains of sugars in chondroitin sulfate (and very similar polymers). Positively charged ions (sodium ions, potassium ions, hydrogen ions, calcium ions are attracted toward negatively charged sulfate ions. They cannot diffuse away because of electrical attraction. Of course, individual sodium ions (etc.) can diffuse away, but there can't be a net loss of positive ions, because every ion that leaves produces an attracting electric field, that pulls in some other positive ion.

    Water diffuses into this area of high concentration of positive ions, just as it would if the increased concentration of these ions were prevented from leaving by a semi-permeable membrane. Therefore you don't need membranes to have an osmotic pressure. Elementary textbooks nearly all say you do, but they are wrong.

    Some other phenomena are also called electroosmosis (because their underlying cause is the same). For example, if you apply an electric voltage to water in a glass tube, you will cause water to flow toward along the glass surface toward the negative electrode. The reason is that positive ions are slightly more concentrated very close to glass surfaces, which is because they release sodium ions when soaked in water. Positive ions get pulled toward the negative electrode, and guess which direction negative ions will be pulled. If you had a polymer that released negative ions, then the electroosmotic water flow would be toward the positive electrode.

    Concrete and bricks are like glass in releasing mostly positive ions when soaked in water. As a result, you can pull water out of brick and concrete walls using electrodes. Although what's being pulled are sodium, potassium and hydrogen ions, water moves along with them; so you might as well be pulling the water out of the concrete etc.

    Another phenomenon called electroosmosis is the production of an electric field by the flow of water through sand, clay, concrete or other materials. This is a perfectly correct use of the word, because the physics is the same - the same as pressure in cartilage, and the same as when water is pulled out of a foundation with electrodes. Not everybody can easily see that all these phenomena result from forces produced by ions held in place by attraction to ionized solids.

    The Wikipedia article gets everything right, except for leaving out cartilage:>

    Unfortunately, Wikipedia's article about cartilage doesn't understand why cartilage swells, and neither do any current textbooks on orthopedics. Scott Gilbert's textbook has no clue that there is anything worth knowing about the physics of cartilage. What sort of evidence would be sufficient to persuade him, or them, is a question worth meditating.

(What about growth pressure? Don't mitoses exert a push? No, apparently not.)


    Contraction of cytoplasmic actin and myosin.

      For example, contraction of primary and secondary mesenchyme cells in urchin blastocoels

    Active bending of epithelial sheets, by acto-myosin contraction of concave surfaces.

      Examples: in-folding of the blastopore in urchins, infolding of neural tubes.


    "Traction" = the shearing force exerted tangentially through plasma membranes of crawling cells.
      Crawling "amoeboid locomotion" of almost all differentiated cell types;
      Not just leucocytes, but also mesenchymal and epithelial cells. All tissue culture cells.

      NOTE: Cell traction has the side-effect of transporting rearward any particle that attaches to a crawling cell.

      This is called "Retrograde Surface Transport" (Filopodia rarely occur, despite textbooks' claims)

      In 1981, Stopak, Wild & Harris proposed a radical new theory that retrograde surface transport of attachments between collagen fibers and mesenchymal cells are (for some cells) the real purpose of traction, the reason why traction is 100 times stronger in some cells than others (that are faster), and the true mechanism that arranges collagen to form ligaments, tendons, fascia and walls of blood vessels ("tunica media") This theory has progressed from "It's crazy!", through "It's impossible!" and some are now saying "We knew it all along!"

      The theory's current respectability owes much to the discovery that culturing stem cells on flexible sheets, like rubber, can control which cell type they differentiate into. i.e. Using a stiff substratum causes differentiation into one cell type, but using a limp or weak substratum causes differentiation into something else. For most developmental biologists, how to control differentiation according to position has long been their holy grail.

      Our citation numbers on the key papers have today reached 940, 682, & 450, for what that's worth (1/17/14)

      Visualize a fleet of many submarines, all traveling along together in the same direction, with their periscopes sticking up through the surface. The submarines represent the moving actin fibers; their periscopes represent the trans-membrane exertion of forces by integrins and fibronectin. Finally, visualize ice-bergs being pushed directionally across the ocean surface by sideways pressure of the the periscopes.

      You can hardly blame people for preferring to imagine that body cells crawl by a cycle of protrusion, attachment, and contraction of filopodia and other outward extensions of advancing cell margins. So what if that is inconsistent with where crawling cells exert forces, and contradicted by rearward transport of particles or any other movable object attached to the outside of the plasma membrane.



      Please read the following excerpt from a review paper by Dr. Louise Cramer, a British Cell Biologist: (The entire paper is available on line, and conveys a (still-up-to-date!) good impression of the difficulties and debates about this subject. It will show you the need and opportunity for more research and careful thinking on this subject.)

      [Frontiers in Bioscience, 2, d260-270, June 1, 1997]


      Louise P. Cramer

      The Randall Institute, Kings College London, 26-29 Drury Lane, London WC2B 5RL, UK.

      The following is a short quote from near the middle of Dr. Cramer's paper:

        "...Early ideas on how actin filaments generated force to drive retrograde particle flow in lamellipodia were theoretical. One quite popular idea was that contraction of an actin filament network moved the lipid bilayer of a lamellipodium backward as a sheet, and structures on the moving sheet rode as passengers (4, 11). At the time this made sense; flow of particles on the surface of lamellipodia were thought to reflect a moving cell surface, and muscle proteins were just beginning to be identified in non-muscle motile cells (reviewed in (12)). This theory was not pursued once Singer and Nicholson (13) introduced the idea that the lipid bilayer was fluid. Of the several alternative explanations offered, the one that turned out to be the most pertinent came from a discussion between Wolpert and Harris in 1973 (11). Wolpert hypothesized that a 'filamentous system' directly moved particles retrograde. Precisely how has been debated since this time. Part of the problem is that over the last 10 years or so different types of particles have been studied in different motile cell types. For example, the tendency has been to view particles flowing retrograde on the cell surface, as the same phenomenon as particles and actin filaments flowing retrograde inside the lamellipodium. It may turn out, however, that retrograde flow of particular types of particles associated with lamellipodia in some motile cell types, may be a separate phenomenon, driven by a distinct mechanism. Perhaps related to this, different results have been obtained in different motile cell types, particularly in Aplysia bag cell neuronal growth cones, fibroblasts and keratocytes. This has led to distinct views on both the mechanism of retrograde particle flow, and function of retrograde actin flow in lamellipodia.

        In this review, I will briefly describe the organization of actin filaments in leading edge structures of adherent, motilearrow to left) and lamella (dashed thick arrow to left), and recently, mostly cells, and in neuronal growth cones. Then, I will describe potential types of actin-dependent motile force to drive retrograde particle flow relative to the substratum in lamellipodia of these cells, and in growth cones of Aplysia neurons. I will present evidence in favor of each type of motile force, and discuss function of retrograde actin flow"...etc.

      I added the boldface and the underlining, for emphasis. They are not in the original paper.


    "Work of Adhesion":

      Malcolm Steinberg and many other good scientists have advocated the theory that at least part of the pulling force by which body cells move is directly caused by formation of new adhesions (either adhesions to other cells, or adhesions to collagen, or adhesions to glass and plastic.

      Other researchers (me, for example) conclude that cells use active (acto-myosin) contractility to pull on adhesions, which creates almost exactly the same result as if the adhesions were exerting the pulling force, because weaker adhesions limit how much force is exerted, so you get a stronger pull when the adhesions are stronger (but not because the process of forming cell adhesions exerts any force, no more than glue attracts objects that it sticks to).

      On the other hand, please don't think that it's impossible for pulling forces to be created by the formation of new molecular bonds. When water is sucked into a towel, the pulling force is created by formation of hydrogen bonds between the water and cloth or paper (or glass). Work of adhesion can occur; it's not impossible or even improbable. The question is: What evidence do we need to prove or disprove it in each given case. It might be that SOME of the pull on cells is work of adhesion, some percentage.

      Movement of cells up adhesion gradients was a major part of the evidence favoring "work of adhesion."

      Most attention to this issue resulted from H. V. Wilson's discovery of sorting out by dissociated and randomly mixed cells of sponges and marine hydroids. (Wilson, himself didn't even believe that cells can sort out; he thought they were re-differentiating.)

      An early hypothesis to explain cell sorting was "selective affinity", which included the idea of each differentiated cell type should have its own special cell-to-cell glue. This is the correct explanation, in my opinion.

      In the 1960s Malcolm Steinberg proposed a clever alternative theory, according to which cell sorting would result from different amounts of only one kind of cell-cell adhesion protein. Part of this theory assumed that pulling forces are exerted by the formation of cell-cell adhesions, so that stronger pulling forces would be exerted by cells that had more adhesion proteins. He called this hypothetical adhesion force the "Reversible Work of Adhesion", and reacted to all critics by claiming that "they didn't understand basic thermodynamics". This intimidated nearly all critics (because, in fact, they really didn't understand thermodynamics, but didn't want to admit it)


Convergence toward particular shapes and arrangements

In addition to finding out which physical forces move cells and collagen around, embryologists need to understand how forces create particular shapes. Embryonic cells somehow "gravitate" to certain geometrical arrangements in preference to others. In order to produce such convergence, there are three requirements: First, the strengths of at least some of the forces need to change as some function of geometrical variables; Second, these variations need to produce exact counter-balances only when the stable geometry exists; And third, all imbalances must be back toward the state of counter-balance.

Many embryologists confuse convergent stability with thermodynamic minimization of free energy. Our textbook makes this mistake on page 72, specifically in relation to reasons why dissociated cells sort out according to germ layer and differentiated cell types. This is an "Emperor's New Clothes" situation. People naturally want to seem wise, and also to trust one another. In this case, the relevant thermodynamic principles are valid only for those special cases in which all the counter-balanced forces in embryos are conservative (don't expend energy when stably counter-balanced) and reversible (don't expend energy when a distortion occurs, from which they then recover.) Forces exerted by springs are conservative and reversible; forces exerted by motors are not. Forces exerted by acto-myosin also are not. Nor are any forces that expend energy. Scott Gilbert doesn't understand this limitation, and wishes to appear wise. It must have been psychologically difficult for him to teach so long at a college where the students are notoriously smarter than most of the faculty.

A better analogy is homeostasis. Our body temperature is held (nearly) constant by counter-balance of shivering (when we get too cold) versus sweating (when we get too warm). This is an over-simplification, but correct in principle. Negative feedback cycles keep many quantitative variables steadily constant, and push these variables back toward their healthy amounts if these variables somehow get distorted. This is the most fundamental concept in Physiology. Luckily, no one succeeded in persuading physiologists that thermodynamics applies to stable counter-balances of active forces. Nobody even tried, that I know of. (But I would greatly appreciate being told about any examples of thermodynamics being mistakenly applied in physiology, or other fields.)

For reasons that I don't understand, nearly everyone assumes that only simple quantitative properties (scalars) can be held constant by negative feedback cycles. Actually, tensor variables and even shapes can be controlled and created by homeostatic mechanisms. This is called "Shape Homeostasis". All you need is counter-balances which change relative strength as functions of shape.

The word "Tensegrity" has also been applied to situations of engineering and bioengineering where counterbalances of tensions versus pressure maintain stability of structures. Buckminster Fuller (real first name Richard) invented the word and popularized this concept, but wasn't born quite soon enough to have invented either tents or suspension bridges. Donald Ingber is currently the major popularizer of applying tensegrity to explanations of cell shapes and tissue structures. (My spell checker still doesn't regard tensegrity as a word, however.) The January 1998 issue of Scientific American magazine explains the foundations of his ideas. He has some on-line videos that may also explain such things; If you find a good one, please tell me the URL.

Tensegrity is highly relevant to orthopedic surgery, and last fall the UNC Biology Dept. had an outstanding seminar on this subject by Dr. Steve Levin,

These are not assigned reading for Biology 441, but are interesting and extremely relevant to biomechanics.


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