Adventures in Multicellularity

The social amoeba (a.k.a. slime molds)

Dictyostelium discoideum

Dictyostelium discoideum

the most studied of the social amoebae / cellular slime molds

predatory soil amoeba that feeds on a variety of microorganisms and decaying matter

(in the lab exist primarily on a diet of E. coli although strains have been selected that grow on complex media alone).

asexually reproducing amoebae (unicellular) under conditions when food is abundant

under starvation conditions a complex developmental cycle is initiated – an adventure in multicellularity.

they have features of both plants and animals• cellulose and development of spores (plant-like)• cell movements involved in morphogenesis (animal-like)

Embryogenesis Dictyostelium Development

Aggregation of many identical cells

Successive divisions of large precursor cell (zygote)

The Natural History of Dictyostelium discoideum

Vegetative Cycle:

Amoeba lives solitary existence feeding on bacteria

Reproduce by binary fission (i.e. divide in half)

Continues as long as food is available

Feeding is by phagocytosis – essentially engulfing their prey with membrane.

RH Kessin (2001)

The Natural History of Dictyostelium discoideum

Chemotaxis and Aggregation:

When the cells begin to starve, they begin a developmental cycle

They produce and release cAMP that acts as a chemoattractant and also as a signaling molecule

The cells comes together (streaming) to form a multicellular tipped aggregate .

This elaborates into a finger or standing slug

RH Kessin (2001)

The Natural History of Dictyostelium discoideum

The standing slug has two fates – it can topple over and become a migrating slug or it can proceed to develop into a fruiting body. The elaboration of the fruiting body begins with culmination

RH Kessin (2001)

The Natural History of Dictyostelium discoideum

Scanning electron micrographs of the different stages of development

RH Kessin (2001)

The Natural History of Dictyostelium discoideum

1.

2.

3.

4.

1. Chemotaxis to signals produced coordinately by the cells (primitive morphogens)

2. Aggregation and cell adhesion (primitive patterning and multicellularity)

3. Differentiation and patterning in the migrating slug and fruiting body (primitive polarity)

4. Competition to be spores (cheating)

RH Kessin (2001)

The Natural History of Dictyostelium discoideum

Enclosed in a sheath (muco-polysaccharide and cellulose)

The tip is a specialized group of cells that control development(remove it and development stops until a new tip is formed)

Under the control of the tip the aggregate elongates and forms the standing slug.

RH Kessin (2001)

The Natural History of Dictyostelium discoideum

The slug typically contains about 100,000 cells and behaves as a single entity. It is capable of both phototaxis and thermotaxis.

RH Kessin (2001)

Dictyostelium aggregates have many properties of an embryo :

• They are essentially a multicellular entity enclosed within a sheath

• They have polarity

• They have exquisite proportioning (1/5 prespore ; 4/5 prestalk)

• They have an organizing center (tip)

• morphogenesis involves cell movements

The essential steps in cell motility

RH Kessin (2001)

Three dimensional reconstruction of a D. discoideum amoebae moving towards a cAMP gradient.

cAMP source

cAMP

Wessel and Soll, Univ. of Iowa

Chemotaxis of single cell towards a point source of cAMP.

The tip of the capillary tube is the source of cAMP. See how quickly the cell responds to changes in concentration. Note the number of pseudopodia protrusions in the amoeba.

Time hr:min:sec:

G. Gerisch, Max Planck Institute, GDR

D. discoideum amoebae chemotaxing toward cAMP

Cells are expressing a GFP-coronin fusion protein: coronin is a cytoskeletal protein that is localized in the extended pseudopods.

G. Gerisch, Max Planck Institute, GDR

Starvation and cAMP signaling

During starvation, cells develop the ability to synthesize, detect and degrade cAMP.

When one cell releases a pulse of cAMP (1), neighboring cells detect it and move up the gradient towards the source (2) .

After a delay of about one minute these cells release cAMP of there own (2) and process gets repeated about every six minutes (3).

This process results in waves of migration (4).

1

2

3

4

RH Kessin (2001)

Aggregation patterns

The patterns of aggregation can be seen by dark-field microscopy because moving and stationary cells reflect light differently – moving cells appear lighter.

Note the waves of cAMP tend to propagate from a central point (‘organizing center’)

RH Kessin (2001)

Dark waves of D. discoideum cells on caffeine agar. Movie constructed from 36 seconds intervals. (F. Siegert and C.J. Weijer J.Cell Sci. 93:325-335 (1989))

Aggregation Patterns

Core of a Spiral Wave in Aggregating D. discoideum cells

10 sec between images

Siegert and Weijer, J. Cell Sci. 93:325-335.

Siegert and Weijer, Curr Biol 1995

Rotation of cells within the mound.

The classical studies of Raper (1940’s)

RH Kessin (2001)

Prestalk and prespore cells do not mix throughout development.

The classical studies of Raper (1940’s)

RH Kessin (2001)

The tip controls events in the slug.

Apical fractions from foreign slugs (B-D) were grafted onto a normal slug. Four independent slugs emerge after ~45 min.

JT Bonner (2000)

Positional Sorting of Prestalk and Prespore cells within the slug

Prestalk cells were grafted onto the anterior end of a slug.

These cells eventually migrate to the proper position at the posterior end of the slug.

JT Bonner (2000)

There is little mixing of prestalk and prespore cells in the slug.

The prestalk cells at the tip move more rapidly in the slug than the pre-spore cells.

Prestalk Prespore

This study used an ingenious method developed by Bonner to generate “two dimensional” slugs. They could contain as few as 50 cells (typical slugs have > 100,000 cells) but the correct proportioning of prestalk and prespore cells was always maintained.

Wolpert, 1998

Movement of Cell Types During Culmination

Weijer, Univ. of Dundee

Note that cells move more rapidly in the tip (prestalk cells) than in the body of the slug (prespore cells). Note also that the tip is further partitioned into Prestalk a nd Prestalk O cells.

Movement of Prestalk Cells in the Slug

Two types of prestalk cells:1) Green (GFP labeled) Prestalk A cells2) Red (red-shifted GFP labeled) Prestalk O cells

Weijer, Univ. of Dundee

Movement of Prestalk Cells During Culmination

Two types of prestalk cells:1) Green (GFP labeled) Prestalk A cells2) Red (red-shifted GFP labeled) Prestalk O cells

Watch closely for the movement of prestalk cells down the core of the culminating slug.

Mound formation can be described by two assumptions: 1.) by considering the population of cells as an excitable medium and 2.) by considering cell movement as the flow of a compressible liquid. The cell movement (fluid flow) is controlled by chemotactic forces dependent on increasing concentrations of the cAMP. The model allows to describe the whole early development from isolated single cells into bifurcating aggregation streams to the formation of a 3-dimensional, hemispherical mound, (see Vasiev B., Siegert F. & C. J.Weijer (1997), J.Theor. Biol. 184, 441-450

A three dimensional hybrid cellular automata/partial differential equation model capable of describing the morphogenesis of Dictyostelium discoideum “from single cells to crawling slug". Using simple local interactions we can achieve the morphogenesis with only three processes: production of and chemotaxis to cAMP and cellular adhesion. The interplay of these processes causes the amoebae to spatially self-organize leading to the complex behaviour of stream and mound formation, cell sorting and slug migration all without any change of parameters. Our model describes the substrate for morphogenesis on which higher cell functions can operate.

Savill and Hogeweg (1997) J. Theor. Biol. 184:0229-235.

http://www-binf.bio.uu.nl/~njs/dicy.html

P. Hogeweg et al Model of Development (Univ. of Utrecht)

Streaming and Mound Formation

Hogeweg Model (cont’d)

• Each amoeba is represented by connected automata in CA system (not just as a single cell)

• Cell-cell adhesion an chemotaxis are represented as simple CA rules

• Amoeba are not rigidly defined by the number of cells but can fluctuate slightly allowing flexible interactions and movements past one another

cAMP waves

Slug development

Hogeweg Model (cont’d)

Yellow PrestalkGreen Prespore

Siegert and Weijer, J. Cell Sci. 93:325-335.

General References:

First Signals : Evolution of Multicellular Development. J.T. Bonner (2000) Princeton University Press

Dictyostelium: Evolution, Cell Biology and the Development of Multicellularity.Richard H. Kessin (2001)Cambridge University Press

Web sites containing numerous links to research sites, resources and databases: http://dicty.cmb.nmu.edu/dicty/dicty.html

http://dictybase.org/dicty.html