Dictyostelium is a member of the Amoebozoa, a taxon that is basal to the Fungi-Metazoa branch.
Introduction to Multicellularity
- A cellular slime mold found on soil and leaves in cool forests.
- Vegetative growth as single-cell amoebae which feed upon bacteria by phagocytosis.
- Development into multicellular organism over 24-hour period.
- Upon starvation, cells aggregate to form mounds of 104-105 cells that organize into a migrating slug with anterior-posterior polarity.
- If no food source is found, slug cells go into culmination/sporulation program and form fruiting bodies.
- Anterior (pre-stalk) cells differentiate into stalk cells that elongate and die.
- Posterior (pre-spore) cells differentiate into spores.
- SEM of Dictyostelium developmental stages. Copyright M.J. Grimson & R.L. Blanton; Biological Sciences Electron Microscopy Laboratory, Texas Tech University
Development of Dictyostelium discoideum poses common questions for development of any multicellular organism:
- How do cells talk to each other?
- How do cells stick together?
- How do cells know which end is up (or forward)?
- How do cells in a multicellular organism form patterns?
Genome: haploid -- easy mutant isolation
- 4 x 107 bp (8 x E. coli genome; 1% of human genome)
- readily transformable, amenable to antisense gene suppression
- homologous recombination for targeted gene knockout or replacement
- genome sequence published in 2005 (Eichinger et al. 2005).
- Cells nearing end of exponential growth (prestarvation) sense accumulation of prestarvation factor (PSF) and conditioned medium factor (CMF) in medium, turn on expression of carA (gene for cAMP receptor CAR1) and acaA (gene for adenylyl cyclase, ACA).
- Starvation causes cells to migrate toward each other, to form mounds of cells. This migration is coordinated, and pulsatile. Cells form small streams that converge toward a central mound. See Dictyostelium early development video.
- Cyclic AMP (cAMP) is the primary signaling molecule that drives aggregation and subsequent development.
- Pulses of cAMP are secreted by aggregating mounds of cells at 6-10 min intervals.
- Cells are chemotactic for cAMP (and for Ca2+).
- CAR1 cell surface receptor for cAMP has 7 transmembrane domains, homologous to rhodopsin.
- CAR1 coupled to G proteins, show adaptation like rhodopsin.
- Adenylate cyclase on membrane is stimulated by CAR1 for positive feedback.
- Secreted cAMP phosphodiesterase (PdsA) hydrolyzes cAMP to attenuate the external cAMP signal.
- CAR1 also stimulates PKA (cAMP-dependent protein kinase) by stimulating ERK2 (MAPKK), which inhibits RegA (cytoplasmic phosphodiesterase).
- PKA inhibits ACA to attenuate external cAMP response, also inhibits ERK2, allows RegA to hydrolyze internal cAMP to restore circuit to resting state.
In migrating slugs, the tip is a source of cAMP
Two new cAMP receptors appear after aggregation: cAR2 and cAR3 (there are a total of 4 different cell surface cAMP receptors, cAR1-4)
- cAR2 is expressed on anterior, pre-stalk cells
- cAR3 is expressed on posterior, pre-spore cells
- cAR4 is essential for normal prestalk/prespore patterning (Ginsburg and Kimmel, 1997)
- Discoidin mediates substrate adhesion, analogous to mammalian fibronectin
- csA (GP80), a lectin-like molecule, has homology to cell adhesion molecules (CAMs) in birds and mammals, and mediates EDTA-stable contacts.
- Targeted disruption of csA gene results in reduction of adhesiveness, but morphogenesis proceeds normally -- csA not necessary for development
- Ectopic expression of csA in vegetative cells causes cells to aggregate and initiate morphogenesis.
- DcCAD-1, a 24-kDa Ca2+-dependent cell-cell adhesion molecule (Sesaki and Siu, 1996).
- Amino acid sequence similar to vertebrate E-cadherin.
- Expressed soon after initiation of development, mRNA peaks at 9h, declines to basal level by 18 h. -- protein level remains constant after maximum at 12 h (Yang et al., 1997).
- Transcription activated by external cAMP pulses, also during vegetative growth in axenic medium (chemically defined, no bacteria).
- Located in cytoplasm initially, relocates to cell periphery before aggregation, enriched on membane ruffles, associated with lamellipodia and filopodia (which form cell-cell contacts).
- Replaced by gp80 in contact regions during aggregation.
- Located primarily at tip and outer margins during chemotactic migration.
- Tiger genes (TgrB1 & TgrC1) cell surface proteins similar to vertebrate MHC proteins (See Science Daily summary of paper by Hirose et al. 2011).
- allelic pairs of TgrB1 and TgrC1 used to discriminate self (genetic identity) vs non-self.
- Cells with self-TgrB1 and foreign TgrC1 do not aggregate.
- Video comparing aggregation of mixtures of compatible and non-compatible amoeba on NY Times
- The anterior 20% of slug cells make stalk, the posterior 80% make spores.
- This proportion is independent of size.
- Halves or thirds of slugs will reform, and if allowed to migrate, form normal fruiting bodies.
- If not allowed to migrate, only the posterior cells will form spores; anterior cells form just stalk.
- Anterior tip of slug has special “organizer” properties -- grafted tips will cause new slugs to split off.
- Diffusible morphogen theory: a concentration gradient of a small molecule provides positional information; threshold concentrations of molecule determine pre-stalk vs pre-spore fate.
DIF -- Differentiation Inducing Factor
- Isolated from slugs using a stalk cell differentiation assay
- Chemical structure reveals small molecule, diffusible in both water and lipids
- Induces stalk cell differentiation, and expression of stalk-cell specific genes
- But concentration gradient of DIF is backwards!! Highest at posterior!!
- DIF sink (DIF-1 dechlorinase) located in anterior tip -- role of DIF metabolites?
- DIF actually appears to induce pre-stalk cell differentiation at early mound stage. Pre-stalk cells then migrate to tip and organize a migrating slug (Early et al., 1995).
- DIF regulates TTGA-binding factor, which binds to direct TTGA repeats to activate transcription of pre-stalk genes (expressed early during mound formation and in tip cells of slug), and binds to inverted TTGA repeats to repress stalk-specific genes (expressed only during culmination, as pre-stalk cells differentiate into stalk cells). The TTGA-binding factors is a STAT protein, containing an SH2 (src homology 2) domain for tyrosine phosphorylation, and a DNA-binding domain (Kawata et al., 1997).
- Mutants unable to synthesize DIF-1 have reduced proportion of pre-stalk cells, and specifically lack basal disc cells and lower cup cells (Saito et al. 2008).
- Continues to be produced after aggregation, proposed to regulate cell sorting, slug migration, cell differentiation and morphogenesis of the fruiting body. Adenylate cyclase (ACA) null mutants do not develop.
- PKA (protein kinase A) is the major downstream transducer of intracellular cAMP signals. Consists of two subunits. PKA-R (regulatory subunit) binds to PKA-C (catalytic subunit) to maintain inactive complex. PKA-R binds cAMP, releases active PKA-C. Mutants in which PKA-R is inactive or PKA-C is constitutively active have accelerated development.
- ACA null mutants (no cAMP made) are rescued almost completely by PKA-C expression (Wang and Kuspa, 1997). They aggregate and develop nearly normal slugs and fruiting bodies, albeit more slowly (30 h vs. 24 h for wild type). Major difference from wild type is that they fail to aggregate at low cell densities, but they do aggregate normally at high cell densities. Therefore, extracellular cAMP is required only for aggregation at low cell densities, not for formation of tip, slug migration, or pre-stalk/pre-spore differentiation. Also, since PKA-C overexpression completely rescues lack of cAMP production, PKA mediates all intracellular cAMP signalling pathways.
- SDF-1 is a phosphorylated peptide that induces pre-spore cells to encapsulate (form spore coat). SDF-1 phosphorylation requires PKA
- SDF-2 is a peptide secreted by pre-stalk cells during culmination, stimulates PKA in pre-spore cells.
- SDF-2 secretion by pre-stalk cells depends on TagC, a pre-stalk specific membrane protease, and feedback stimulated by low levels of external SDF-2 via DhkA-mediated inhibition of RegA and stimulation of PKA.
- Pre-spore cells respond to SDF-2 via DhkA, a receptor histidine kinase homologous to bacterial two-component sensor histidine kinases, which inhibits RegA to activate PKA.
Chen & Schaap (2012) reported evidence that cyclic di-(3′:5′)-guanosine monophosphate (c-di-GMP) is the morphogen responsible for stalk cell differentiation. The diguanylate cyclase gene (DgcA) is expressed at the tip of the slug. DcgA knockouts remain in the slug stage and fail to form fruiting bodies. No stalk-specific genes are expressed in these mutants. The mutant phenotype is rescued by addition of c-di-GMP.
Dictyostelium: 3 life cycles, 3 sexes!
Dictyostelium discoideum has 3 sexes, or mating types, and the molecular basis of sex-determination has only recently been characterized (Bloomfield et al. 2010). See recent blog: Dictyostelium discoideum is pretty cool:
Bloomfield, G, J Skelton, A Ivens, Y Tanaka, RR Kay, 2010. Sex determination in the social amoeba Dictyostelium discoideum, Science 330:1533-1536 http://dx.doi.org/ 10.1126/science.1197423
Brown, J.M. and R.A. Firtel, 1999. Regulation of cell-fate determination in Dictyostelium
Chen, Z. and P. Schaap, 2012. The prokaryote messenger c-di-GMP triggers stalk cell differentiation in Dictyostelium, Nature 488:680-683
Devreotes, P., 1989. Dictyostelium discoideum: A model system for cell-cell interactions in development, Science 245:1054-1058.
Early, A., T. Abe and J. Williams, 1995. Evidence for positional differentiation of prestalk cells and for a morphogenetic gradient in Dictyostelium, Cell 83:91-99.
Eichinger L, et al. 2005 The genome of the social amoeba Dictyostelium discoideum, Nature 435(7038): 43-57. DOI:10.1038/nature03481
Faix et al., 1990. Constitutive overexpression of the contact site A glycoprotein enables growth- phase cells of Dictyostelium discoideum to aggregate, EMBO Journal 9:2709-2716.
Ginsburg, G.T. and A.R. Kimmel, 1997. Autonomous and nonautonomous regulation of axis formation by antagonistic signalling via 7-span cAMP receptors and GSK3 in Dictyostelium. Genes Dev. 11:2112-2123.
Gura, T., 1997. One molecule orchestrates amoebae, Science 277:182.
Hirose S, R Benabentos H-I Ho, A Kuspa, G Shaulsky 2011 Self-Recognition in Social Amoebae Is Mediated by Allelic Pairs of Tiger Genes Science DOI: 10.1126/science.1203903
Jermyn et al., 1989. A new anatomy of the prestalk zone in Dictyostelium, Nature 340:144-146.
Kawata, T., A. Shevchenko, M. Fukuzawa, K.A. Jermyn, N.F. Totty, N.V. Zhukovskaya, A.E. Sterling, M. Mann and J.G. Williams, 1997. SH2 signaling in a lower eukaryote: a STAT protein that regulates stalk cell differentiation in Dictyostelium, Cell 89:909-916.
Kay et al., 1993. A localized differentiation-inducing-factor sink in the front of the Dictyostelium slug, Proc. Natl. Acad. Sci. USA 90:487-491.
Klein et al., 1988. A chemoattractant receptor controls development in Dictyostelium discoideum, Science 241:1467-1472.
Morris et al., 1987. Chemical structure of the morphogen differentiation inducing factor from Dictyostelium discoideum, Nature 328:811-814.
Saito, T, A Kato and RR Kay, 2008. DIF-1 induces the basal disc of the Dictyostelium fruiting body, Dev. Biol. 317:444-453 doi: 10.1016/j.ydbio.2008.02.036
Schaap, P., 2011. Evolutionary crossroads in developmental biology: Dictyostelium discoideum, Development 138:387-396 doi: 10.1242/dev.048934
Schnitzler, G.R., C. Briscoe, J.M. Brown and R.A. Firtel, 1995. Serpentine cAMP receptors may act through a G protein-independent pathway to induce postaggregative development in Dictyostelium, Cell 81:737-745.
Sesaki, H. and C.H. Siu, 1996. Novel redistribution of the Ca(2+)-dependent cell adhesion molecule DcCAD-1 during development of Dictyostelium discoideum. Dev. Biol. 177:504-516.
Soderbom, F. and W.F. Loomis, 1998. Cell-cell signaling during Dictyostelium development. (review) Trends Microbiol. 6:402-406.
Wang, B. and A. Kuspa, 1997. Dictyostelium development in the absence of cAMP. Science 277:251-254.
Yang, C., S.K. Brar, L. Desbarats and C.H. Siu, 1997. Synthesis of the Ca(2+)-dependent cell adhesion molecule DdCAD-1 is regulated by multiple factors during Dictyostelium development, Differentiation 61:275-284.
Yu, Y. and C.L. Sax III, 1996. Differential distribution of cAMP receptors cAR2 and cAR3 during Dictyostelium development. Dev. Biol. 173:353-356.
Check out the following Web sites!
http://dictybase.org/index.html Dictyostelium database. Links to other Dictyostelium laboratories and resources worldwide.