Unsolved Problems in Cell BiologyBiology 446Sept 25, 2015Albert Harris

What Can Be Learned About Normal Embryonic Development From Studying Sorting-out of Dissociated and Randomly Rearranged (& Already Differentiated) Cells?

What Forces Cause Sorting-out of Dissociated and Rearranged Cells?
Selective Cell-Cell Adhesion Proteins (Cadherins, N-Cam, etc.)

1907: H.V. Wilson discovers sorting out by dissociated and randomly mixed sponge cells. (He concludes that cells switch differentiated cell types, or develop from archeocytes = stem cells) Wilson, H.V. (1907). On some phenomena of coalescence and regeneration in sponges, J. Exp. Zool. 5, 245-258

1930s-50s Johannes Holtfreter discovers frog & salamander embryo cells sort out by germ layer (ectoderm, mesoderm, endoderm; and subdivisions of them. Hypothesizes that differences in "tissue affinity " cause sorting. (& not stem cells or changes in cell differentiation). But does affinity equal adhesiveness?

1955 Townes and Holtfreter publish their classic paper on cell sorting: Townes' 9 years of PhD research.

Townes, P.L., and Holtfreter, J. (1955).Directed movements and selective adhesion of embryonic amphibian cells Journal of Experimental Zoology 128, 53-120.

1950s Aaron Moscona and John P. Trinkaus (separately) demonstrate that bird and mammal embryo cells can dissociated with proteolytic enzymes and EDTA (divalent ion chelator), and that they sort out according to differentiated cell type (heart, liver, pigmented retina) with no changing of cell type, producing spheres of one cell type surrounded by cells of another cell type.

Trinkaus, J.P., and Groves, P.W. (1955). Differentiation in culture of mixed aggregates of dissociated tissue cells. Proc. Natl. Acad. Sci. USA 41, 787-795.

Trinkaus, J.P., and Lentz, J.P. (1964). Direct observation of type-specific segregation in mixed cell aggregates. Developmental Biology 9, 115-136.

Moscona tries to discover tissue-specific cell-cell adhesion proteins (like cadherins), but his bioassays are not specific enough. Trinkaus decides that sorting out is unimportant because it doesn't happen in normal embryos.

1963 Malcolm Steinberg invents a way to explain sorting out based on differences in amount of adhesiveness: The "Differential Adhesion Hypothesis" sometimes called the "Thermodynamic Theory".

Steinberg, M. (1963). Reconstruction of tissues by dissociated cells. Science 141, 401-408. (& many later papers)

1969 Herbert Phillips compares resistance to centrifugal flattening of spheres of different cell types, & claims this is a way to measure cell-cell "work of adhesion"

Phillips, H.M., and Steinberg, M.S. (1969). Equilibrium measurements of embryonic chick cell adhesiveness. I. Shape equilibrium in centrifugal fields. Proc. Natl. Acad. Sci. USA 64, 121-127

Forgacs, G., Foty, R.A., Shafrir, Y., and Steinberg, M.S. (1998). Viscoelastic properties of living embryonic tissues: a quantitative study. Biophysical Journal 74,2227-2234.

1970 Steinberg compares inside-outside locations of sorting for about 8 differentiated cell types

Steinberg, M.S. (1970). Does differential adhesion govern self-assembly processes in histogenesis? Equilibrium configurations and the emergence of a hierarchy among populations of embryonic cells. Journal of Experimental Zoology 173, 395-433.

All comparisons of cell types obey "transitive hierarchy"= if A inside B & B inside C: then A inside C (This was predicted by his DAH theory.)

Meanwhile, (1970s & 80s) Gerald Edelman, M. Takiechi and others are discovering different kinds of cell-cell adhesion proteins.

Thiery, J.P., Duband, J.L., Rutishauser, U., and Edelman, G.M. (1982). Cell adhesion molecules in early chicken embryogenesis. Proc. Natl. Acad. Sci. USA 79, 6737-6741.

1976 Critique of Differential Adhesion Hypothesis: Proposal by Harris that Steinberg have been measuring cell contractility, instead of adhesiveness.

Harris, A.K. (1976). Is cell sorting caused by differences in the work of intercellular adhesion? A critique of the Steinberg hypothesis. Journal of Theoretical Biology 61, 267-285.

1994 Steinberg and Takeichi insert genes for P-Cadherin (and E-Cadherin) into cells of a tissue culture line

Steinberg, M.S., and Takeichi, M. (1994). Experimental specification of cell sorting, tissue spreading, and specific spatial patterning by quantitative differences in cadherin expression. Proc. Natl. Acad. Sci. USA 91, 206-209.

Those cells having the most cadherin sort out into internal spheres, relative to cells with less cadherin.
Also see this article from 1988.

2002 Wayne Brodland proposes a Differential Interfacial Tension Hypothesis". He also has a computer simulation on line. And a good paper about "Finite Element" computer simulations of epithelia and other living tissues.

2005 Foty and Steinberg summarize the evidence from their point of view, which is that if differences in amounts of cadherins can cause sorting out.
Foty, R.A., and Steinberg, M.S. (2005). The differential adhesion hypothesis: a direct evaluation. Developmental Biology 278, 255-263. Another interesting paper about the thermodynamics of cell adhesion: by Bell and Dembo.
Bell, G.I., Dembo, M., and Bongrand, P. (1984). Cell adhesion. Competition between nonspecific repulsion and specific bonding. Biophysical Journal 45, 1051-1064

The following paper showed that cancerous cells sort out (to the exterior position) relative to equivalent non-cancerous cells; Does this mean the cancerous cells are less adhesive, or exert weaker contractility?)
Gershman, H., Drumm, J., and Culp, L. (1976). Sorting out of normal and virus-transformed cells in cellular aggregates. Journal of Cell Biology 68, 276-286

2008 C-P Heisenberg (Krieg et al.)
Krieg, M., Arboleda-Estudillo, Y., Puech, P.-H., Käfer, J., Graner, F., Müller, and Heisenberg, C.-P. (2008). Tensile forces govern germ-layer organization in zebrafish. Nature Cell Biology 10, 436 (2008)

Part of which says "Cell-cortex tension Tc can then be calculated using the following equation

(F = force, = indentation, Rc = cell radius and Rb = bead radius)"

Students who took Bio 441 (Vertebrate Embryology) last spring learned what false assumption is made by this equation. (Tension is not allowed to vary as a function of direction, or to vary with location)

"Jeremy B. A. Green (2008) Sophistications of cell sorting. Nature Cell Biology 10, 375-377

The self-sorting of early embryonic cells is mediated not only by pure differential adhesion but also involves other processes. Direct force measurements reveal the role of cell-cortical tension, whereas epithelial-wrapping dominates the sorting of enclosed mesenchymal cells.

To some the DAH was only one of the possible mechanisms and, despite acceptance as the conventional wisdom, DAH has remained controversial.

Apart from requiring a change in what textbooks say about the importance of differential adhesion in cell self-sorting, the work of Krieg et al. leads us to reconsider the role of different cell-biological processes in self-sorting. For a start, the cortical actomyosin cytoskeleton becomes more significant.

The exciting aspect of these papers is that, building on the brilliance of Holtfreter, Steinberg, and others, they represent a new chapter in the analysis of morphogenesis, in which high-resolution force measurement and molecular analysis will be combined with more physiological and multi-component models. Thanks to this type of analysis, the complexity of real embryos is gradually coming within the grasp of hardcore cell biology."

Amack, J.D., and Manning, M.L. (2012). Knowing the boundaries: extending the differential adhesion hypothesis in embryonic cell sorting. Science 338, 212-215

Manning, M.L., Foty, R.A., Steinberg, M.S., and Schoetz, E.-M. (2010). Coaction of intercellular adhesion and cortical tension specifies tissue surface tension. Proc. Natl. Acad. Sci. USA 107, 12517-12522

Manning, M.L., Foty, R.A., Steinberg, M.S., and Schoetz, E.-M. (2011). Reply to Krens et al: Cell stretching may initiate cell differentiation. Proc. Natl. Acad. Sci. USA 108(3):E11-E11.


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