Biology 466    Unsolved Problems Fall 2010

Outline history of cell sorting discoveries and debates


H. V. Wilson (of UNC) notices that living sponge tissues become disorganized when in poor culture conditions, but can reorganize if conditions are improved. (Plus, he must have known about gemmules in fresh water sponges)

Wilson tries mixing physically dissociated cells from different species of salt water sponges; (Explicitly hoping to make chimeric sponges that could be economically useful) But discovers rapid sorting-out by species, into separate masses of cells.

Julian Huxley repeats sponge dissociation, using kinds of sponges that have larger cells, that are easier to watch and distinguish. He proposes that rearrangement according to differentiated cell type is the means by which functional individuals are re-formed.

Wilson dissociated cells of soft corals (Phylum Cnideria), and these also re-form functional organisms.
In this paper, Wilson concludes that it is more likely that cells are changing from one differentiated cell type to another, instead of rearranging (contrary to Huxley's proposal).

Wilson later decides that maybe the new sponges are formed by cells that had previously not been differentiated (archeocytes) and publishes papers describing cells that seem to be in intermediate stages of (re?) differentiation.

Hans Holtfreter uses slightly acidic and low calcium water to cause early embryos of salamanders and frogs to separate into their individual cells, which he then mixes randomly. In contrast to what happened in sponges, cells of different species of amphibian DO NOT sort out from each other; therefore, their color differences can be used to track cell fates.

Holtreter (chemically) dissociates amphibian cells of mesoderm, endoderm, neural ectoderm, skin ectoderm, and other embryonic subdivisions, mixes different combinations of these into random mixtures, and discovers that cells sort out by "germ layer" (for example, ectoderm sorts out from ectoderm, and skin ectoderm sorts out from neural ectoderm), but that instead of sorting into different piles of cells (as did cells of different species of sponges), the amphibian cells sort out with one kind of cell forming an inner core, surrounded by cells of the other kind of cell.

Holtfreter concludes that sorting is caused by differences in "tissue affinity", by which he meant to include not just selective adhesiveness but also differences in chemotaxis, or any other behavior or property that could rearrange cells. But almost everyone interprets his paper to have proposed differences in adhesiveness, only. He resented that.

Townes and Holtfreter publish a classic (long!) paper in J. Experimental Zoology, with a few photographs, and lots of drawings of what happens for every different random mixture of dissociated amphibian cells of different germ layers and other embryonic subdivisions. This was Townes MD/PhD thesis research, and took him nine years to do!

Aaron Moscona discovers that bird and mammal embryos can be dissociated into single cells (without killing the cells) by soaking them in solutions of proteolytic enzymes, and also chelators like EDTA. The motivating assumption was that cells were held together by "calcium bridges" and by selective adhesion proteins (that get cut by the enzymes).

Moscona mixes heart cells from chick embryos and liver cells from mouse embryos, puts them in flasks on shaker tables. First they aggregated into random mixtures of heart and liver cells; and then these cells rearranged to form interior masses of heart cells, within a surrounding continuum of liver cells. For any combination of differentiated cells, one cell type would always sort out "interior", and the other cell type would sort out "exterior".

Moscona concludes that each differentiated cell type most have its own special kind of cell-cell adhesion protein. This seems to have been basically correct, but Moscona was not able to identify the actual proteins, because his bioassay methods were not specific enough. (Anything sticky gave a positive result, and caused larger aggregates, sooner!)

Other scientists, and especially Takiechi in Japan, succeed in identifying the selective cell-cell adhesion proteins, one main family of which are named "cadherins".

Malcolm Steinberg proposed an alternative explanation for cell sorting, by means of differences in amount of cell-cell adhesion. (The "Differential Adhesion Hypothesis", often called the "DAH"). Instead of having different kinds of adhesion protein, each differentiated cell type would have a different amount of adhesion, specifically a different "reversible work of adhesion". This is a concept borrowed from thermodynamics, in which "work" means some transfer or effect of energy. The DAH has often been called the "Thermodynamic Theory" of cell sorting.

Adam Curtis (in Scotland) proposed a timing theory, according to which different cell types recover from dissociation (and the proteolytic enzymes) at different rates. The cell types that recover soonest would sort out to the more internal location, relative to cell types that recovered more slowly.

John Phillip Trinkaus used tritiated thymidine to label embryonic cells, and autoradiography to distinguish labeled cells from unlabeled cells in masses of reaggregating chicken embryo cells, of various differentiated cell types. He discovered that differentiated cells never switched from being one cell type to another cell type, and that they always rearranged according to cell type, with one cell type forming interior clusters and the other cell type becoming continuous and exterior.

He confirmed this by dissociating pigmented retina cells, and randomly mixing these with other cell types, and making time lapse films of the mixtures of cells as they sorted out. The color of the pigment cells made them easily visible. But he had been hoping that the cells would change from one differentiated cell type to another, and wanted to study the mechanisms that could cause this re-differentiation according to cell locations.

When I was a college freshman, I heard a lecture about these different cell sorting observations, and decided that the embryonic mechanisms that put differentiated cells in their correct locations were probably also the cause of cell sorting, and that the best way to discover the mechanisms that form tissues would be to study cell sorting. After I graduated, I asked Prof Trinkaus if I could come to his laboratory and try to prove this using sea-squirt cells(!). He arranged for me to become a graduate student, and to work in his lab, but insisted I study gene expression in fish embryos, and then learn transmission electron microscopy and use it to study the structure of cell-cell adhesions.. While in his lab, at least 70% of my effort was spent studying sorting out by chicken cells, & time lapse films and electron micrographs of sea squirts.

As a college senior, I had a choice between taking embryology and taking thermodynamics. I took the latter, and for embryology I just bought the textbook and read it after graduation.

Steinberg has made many experiments designed to confirm his DAH. In one set of experiments, he dissociated many different tissues (for example, pigmented retina, epidermis, heart, cartilage etc.) and combined these in pairs, having made the prediction that whenever cell type A sorted out to an internal position relative to cell type B, then if cell type B sorted out to an internal position relative to cell type C, then in mixtures of cell type A and C, it would always be true that cell type A would sort out to the more interior position relative to cell type C. This prediction is based on the idea that if A is more adhesive than B, and B is more adhesive that C, then A would always be more adhesive than C. After trying forty five different combinations of different pairs of cell types, and ALWAYS finding that the prediction was confirmed, nobody can blame the man for being annoyed by the criticism (that I published) that the same prediction would have been made based on any theory that explained inside-outside sorting positions on the basis of any quantitative variable. Part of my criticism was that there were other quantitative variables, that would have made this same prediction, so that the results of the 45 pair-wise combinations was evidence that some quantitative variable was responsible, but that this quantitative variable could just as well be strength of contractility.

Anyway, by that time cadherins were beginning to be discovered (although that name wasn't invented until later); so it was clear that adhesiveness was a qualitative variable (differing in kind, rather than amount, between cell types). My idea was that the part of cell sorting where like bunches together with like is caused by them having different cadherins; a but that the later parts of cell sorting, during which cells of one type go to the interior and others go to the exterior, are produced by quantitative differences in contractility of cell surfaces, especially in areas where cells touch the surrounding media, or where they touch cells of a different cell type. Remember, I had been watching cells sort out for years, when I was doing my PhD thesis research on different subjects. Also, based on having taken the regular chemists course in thermodynamics, I could see that it was bogus to assume that cell-cell adhesion was an energetically reversible process, and the DAH assumed that no active forces (like acto-myosin contraction) were contributing to cell rearrangement.

Steinberg and his graduate student Herbert Phillips did some excellent experiments in which centrifugal force was used to flatten multicellular aggregates of particular differentiated cell types. They discovered that the degree of flattening produced by a particular cell type varied according to the same "hierarchy" of internal versus external sorting out. Specifically, those cell types that sort out to the more internal position will flatten less than aggregates of a cell type that would sort out to a more external position. They already believed that the resistance of cell aggregates to flattening was caused by the energy change of pulling apart cell-cell adhesions. Therefore, the results were exactly as they had predicted. The stronger the cell-cell adhesion (which they would call the "reversible work of adhesion"), the greater the tendency to sort out to an internal position, relative to some cell type with a smaller "reversible work of adhesion". They believed that there was some thermodynamic law that would guarantee that such a variable would have to exist. My understanding of the subject is that adhesion need not be energetically reversible, and also need not exert any pulling force on cell locations. Cells definitely use adhesions to pull themselves around, but I believe it's still an open question whether the actual process of forming adhesions ever exerts a pulling force, like magnets attracting each other. Part of my actual thesis work was about formation of adhesions between cells and different plastics, metals and glass, and whether formation of adhesion pulls cell edges. Two reasons to conclude adhesion doesn't pull are * the cell margins extended forward 5 microns or more beyond the most anterior adhesions the plastic, and ** the cell-substratum adhesions covered only a small fraction (<10%) of the area of cell-substratum contact. Although spreading tissue culture cells look as if they are maximizing their adhesions to the glass or other materials they crawl on, really they aren't. But they sure can look like they are!

My hypothesis has been that cell aggregates round up because the parts of cells at the surface contract more strongly than in the interior, so they round up like a balloon, and that the tension forces are caused by acto-myosin contraction. Of course, the cells have to stick to each other; and anyway, one can easily see that cells do stick to each other. The real question is, when spherical masses of one cell type resist flattening more than masses of some other cell type, is this because of stronger contractions of the surfaces, or is it because of stronger adhesions between the cells. This is a factual question. In principle, either could be true, and either could produce almost indistinguishable shape changes. Some mixture of effects could also occur, and be difficult to distinguish which is the driving force. Meanwhile, I think Phillips and Steinberg were actually measuring differences in active contractility of cells at the surfaces of their aggregates, whereas they think they were measuring forces of cell-cell adhesion.

On the other hand, they and I are almost the only scientists who believe that gastrulation, neurulation and other morphogenetic cell movements, in developing embryos, are caused by differences in the physical forces that they measured using centrifugal flattening, and that I measured using rubber substrata. Holtfreter and Trinkaus thought we were all crazy to worry so much about theories and thermodynamics, and nearly all embryologists agree with them. The consensus is that Wilson's research led eventually to the discovery of cadherins (which is true!), but that's its only significance.

My former PhD student Calhoun Bond (now a professor at Greensboro College) and I made time lapse films and videos of living intact sponges, and we discovered that their differentiated cells rearrange all the time, not just after somebody dissociates them. I think this supports the idea that cell sorting has the same mechanism as normal pattern formation inside the body.


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