DB3002 Dictyostelium and Arabidopsis

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DB3002 Dictyostelium and Arabidopsis

• J Martin Collinson
• School of Medical Sciences
• University of Aberdeen
• m.collinson_at_abdn.ac.uk
• Tel F55750

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Dictyostelium discoideum

• A soil-living slime mould (social amoeba)
• Neither a true fungus nor a plant
• Slime moulds are normally regarded as a
  Kingdom-level grouping, somewhere between
  animals and fungi.
• Main uses
• - genetic and pharmacological study of cell
  migration
• - study of simple patterning and cell-cell
  interaction during morphogenesis

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The life cycle of Dictyostelium
Takes about a day from starvation to fruiting
body. Myxamoebae are haploid and the entire life
cycle is completed in the haploid state. Fusion
of myxamoebae (fertilisation and a sexual stage
is much rarer)
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The embryology of Dictyostelium
To aggregate properly, myxamoebae need to produce
cell surface adhesion molecules e.g. DdCad1 Not
all cells in the slug are equal those at the
front are mostly going to be stalk, and those
towards the back will be primarily spores.
There is a lot of cell movement and sorting
within the slug. Pre-spore and pre-stalk cells
express different genes
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The embryology of Dictyostelium
Although in theory any of the myxamoebae single
cells are capable of forming stalk or spore, in
practice they are biased to form one or the
other. Cell cycle stage counts. When starvation
occurs, those cells in S phase or early G2 have
high calcium levels and tend to become stalk.
Those in mid-late G2 have lower calcium levels
and tend to become spores.
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The embryology of Dictyostelium
Its development is regulative If you chop the
front end off the grex, cells that would normally
have become spores are respecified to form
stalk. This ability to compensate when something
goes wrong during development is called
regulation
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Dictyostelium as a model of chemotactic cell
migration
Cyclic AMP cAMP is the critical signalling
molecule. Dictyostelium cells migrate up a
gradient of cAMP.
Real-time speed would be 20 mm/min
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Dictyostelium as a model of chemotactic cell
migration
Fruiting body formation is a response to
starvation. Starving myxamoebae release cAMP
all myxamoebae are more-or-less equally capable
of doing this (i.e. no dominant cells necessarily
pulling all the others). Cells will, on average,
move towards the areas where most other cells are
starving i.e. where most cAMP is being
released.
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Dictyostelium as a model of chemotactic cell
migration
cAMP release and migration occurs in
pulses. Cells receive cAMP, and move up the
gradient towards the source for a minute or so.
They also produce cAMP of their own They then
stop and become unresponsive to further cAMP for
several minutes and the receptors are cleared of
cAMP by a phosphodiesterase. These pulses of
cAMP production and responsiveness pull cells in
waves into apparently coordinated streams that
converge together to form the aggregate blob that
becomes the grex. Migration in response to cAMP
pulse changes cytoskeleton and hence cell shape,
and with fancy microscopy you can use this to
see the spiral waves of cAMP travelling through
the colony. Genetic mutations of Dictyostelium,
e.g. Gb- (lacks a G-protein component of
intracellular signaling), that lack chemotactic
response allow you to genetically dissect the
molecular pathways underlying how cAMP reception
causes cell migration.
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Dictyostelium as a model of chemotactic cell
migration
Phase contrast microscopy of Dictyostelium
aggregates visualises cAMP waves
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Dictyostelium as a model of chemotactic cell
migration
Other videos on the web Some saddo is
sticking Dicty on YouTube. http//uk.youtube.com
/watch?vuqi_WTllG7ANR1 shows a starving field
of cells turning into slugs, with spiralling
waves visible. http//uk.youtube.com/watch?vhpHp
BHJZQvU shows myxamoebae pulsing and aggregating,
with the final point of aggregation suddenly
appearing. Videos shown here came from
http//jcs.biologists.org/cgi/content/full/114/1
3/2513/DC1 And the fantastic Dictyostelium
Cinema (grab your popcorn, needs
QuickTime) http//www-biology.ucsd.edu/firtel/mo
vies.html
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Dictyostelium as a model organism

• Why is this all for fun or do we learn
  something useful?
• Study of tissue differentiation and organ
  formation in a very basic system an organism
  that is only multicellular in a part-time way.
  May tell us things about how multicellular
  animals first developed/evolved.
• Genetic mutants of Dictyostelium have been
  developed, and it is possible to
  pharmacologically disrupt them.
• The signalling pathways used during cell
  polarisation and migration in Dictyostelium are
  very substantially the same as those in higher
  eukaryotic cells, including mammalian. E.g. PI3
  kinase at front, PTEN at back, in Dicty as in
  migrating neutrophils.
• http//uk.youtube.com/watch?vYoXWbr45rsQfeature
  related
• http//uk.youtube.com/watch?vZUUfdP87Ssg
• Hence study of Dictyostelium (relatively simple)
  provides information about higher (more
  complicated) cell behaviour in multicellular
  organisms. Widely used as a genetic model.
• http//www.lifesci.dundee.ac.uk/people/jeff_willia
  ms/research/

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Plant developmental biology

• Not something you think about much, but essential
  for e.g. crop breeding, GM plants.
• Fundamental differences from animal biology
• Plant cells do not normally migrate - encased in
  cellulose.
• Alternation of generations diploid and haploid
  multicellular stages.
• Extreme developmental plasticity
• Independent evolution of developmental
  mechanisms.
• - e.g. developmental patterning Hox genes in
  animals, MADS genes in plants.

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Plant developmental biology

• Most important to remember that embryology can
  occur through the life of the plant.
• Meristems the growing tips at the top (flower
  meristems, shoot meristems) or bottom (root
  meristems) of the plant.
• these are where the developmental patterning
  happens.
• Plants can be visualised as adult organisms
  surrounded by a cloud of embryos (meristems)
• at their tips.

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Meristems
A relatively small number of undifferentiated,
proliferating stem cells (or mother cells) give
rise to cells which have to differentiate
appropriately. Most cell division occurs in
meristem, with subsequent growth being by cell
enlargement. Shoots meristems more complicated
than root, because they have to give rise to an
orderly arrangement of side shoots (axillary bud
meristems) requires cell signalling. Close
proximity of one meristem inhibits others.
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Meristems
Meristems need to know their identity (root,
shoot, flower) and to compartmentalise properly
within this (petals, sepals, stamens, carpels).
This is down to tissue-specific expression of
important developmental genes.
Expression of SERRATE genes in apical meristem
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Meristems
Cells in different layers of the meristem (L1,
L2, L3) tend to contribute to different tissues.
Normally L1 (epidermis) produces no chlorophyll
but L2 and L3 do. If one of the L2 or L3 layers
is not producing chlorophyll, you get variegated
plants.
L3 is green, L2 not, in this plant
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Arabidopsis thaliana
A model plant system. Pan-Palearctic
weed. Rapid life cycle (as little as 6
weeks). Small genome 160 Mbp, fully
sequenced. 27000 genes Can do genetic
crosses. Can make genetically modified plants by
infecting them, with cultures of Agrobacterium
tumefaciens a pathogen that integrates some of
its own DNA - the T-DNA into its hosts genome.
You can modify the T-DNA to include a transgene,
and that transgene will be integrated. (Other
genetic plant models tobacco and maize).
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Genetics of flower patterning
Organised in 4 rings (whorls)
4. sepals
1. Carpels (female)
2. Stamens (male)
3. petals
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Genetics of flower patterning
Wild-type Arabidopsis flowers (top left) and
genetic mutants affecting flower formation.
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Genetics of flower patterning
There are three classes of flower-patterning
genes A, B and C. The combinatorial expression
of these genes in different spatial regions of
the floral meristem determines the identities of
the different organs of the flower. If all genes
are missing, all parts of the flower turn into
leaves.
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Genetics of flower patterning
There are three classes of flower-patterning
genes A, B and C. The combinatorial expression
of these genes in different spatial regions of
the floral meristem determines the identities of
the different organs of the flower. If all genes
are missing, all parts of the flower turn into
leaves.
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Genetics of flower patterning
The classes A, B, C genes have been well
characterised. Class A e.g. APETALA2 Class B
e.g. PISTILLATA Class C e.g AGAMOUS Are
mostly DNA-binding transcription factors of the
MADS-box family. They are homeotic genes
(turning one part of plant into another
part). Vertebrate homeotic genes are largely
homeobox-containing transcription factors. MADS
and Hox genes present in unicellular organisms.
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Genetics of flower patterning
There are three classes of flower-patterning
genes A, B and C. Epistatic to the ABC class
genes, are genes such a SEPELLATA. Mutation in
SEPALLATA means flower are whorls of sepals
only. Ectopic expression of SEPALLATA in a leaf
turns it into a petal. SEPALLATA is also a
MADS-box transcription factor.
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Summary

• Dictyostelium
• A social amoeba with a well characterised
  chemotactic cell migration that acts as a model
  for cell migration during development of
  vertebrate embryos.
• Also a useful model for development of new drugs
  against protozoan parasite
• Arabidopsis
• Genetically accessible plant model central to
  understanding how plant development is regulated.
  
• Plant developmental pathways diverged from
  animal very early in evolution, and although some
  of the principles are the same (e.g. homeosis),
  the genes involved may be from different
  families.