The Amoebozoa

Abstract

The model organism Dictyostelium discoideum is a member of the Amoebozoa, one of the six major -divisions of eukaryotes. Amoebozoa comprise a wide variety of amoeboid and flagellate organisms with single cells measuring from 5 μm to several meters across. They have adopted many different life styles and sexual behaviors and can live in all but the most extreme environments. This chapter provides an overview of Amoebozoan diversity and compares roads towards multicellularity within the Amoebozoa with inventions of multicellularity in other protist divisions. The chapter closes with a scenario for the evolution of Dictyostelid multicellularity from an Amoebozoan stress response.

Full text

1
Ludwig Eichinger and Francisco Rivero (eds.), Dictyostelium discoideum Protocols, Methods in Molecular Biology 983,
DOI 10.1007/978-1-62703-302-2_1, © Springer Science+Business Media, LLC 2013
Chapter 1
The Amoebozoa
Christina Schilde and Pauline Schaap
Abstract
The model organism Dictyostelium discoideum is a member of the Amoebozoa, one of the six major
divisions of eukaryotes. Amoebozoa comprise a wide variety of amoeboid and fl agellate organisms with
single cells measuring from 5 μ m to several meters across. They have adopted many different life styles and
sexual behaviors and can live in all but the most extreme environments. This chapter provides an overview
of Amoebozoan diversity and compares roads towards multicellularity within the Amoebozoa with inven-
tions of multicellularity in other protist divisions. The chapter closes with a scenario for the evolution of
Dictyostelid multicellularity from an Amoebozoan stress response.
Key words Amoebozoa , Protista , Aggregative multicellularity , Encystation , Sporulation ,
Morphogenesis , Cyclic AMP signaling , Phylogeny
The Dictyostelids have fascinated biologists for over 150 years with
their ability to assemble up to a million amoebas into a tactile
migrating organism, which, after seeking out a site for spore dis-
persal, transforms into a well-balanced fruiting structure. The
development of a range of molecular genetic and cell biological
procedures for the species Dictyostelium discoideum over the past
30 years has established this species as an important model organ-
ism for the study of fundamental cell biological and developmental
processes (
1 ) . More recently, the evolution of social behavior and
the study of genes associated with human diseases and bacterial
infections have been added to the repertoire of research questions
that can be addressed in Dictyostelia (
2 ) .
With putative applications of research in mind, fi ndings obtained
in D . discoideum are usually extrapolated to and compared with
research in higher vertebrates. However, we should not lose sight of
the fact that D . discoideum is evolutionary very distant from Metazoa,
including vertebrates, and represents an independent invention of
1 Introduction

2 Christina Schilde and Pauline Schaap
multicellularity in an entirely different eukaryotic lineage ( 3 ) . To
understand and recognize the core components of any process under
study and to separate these components from species- or clade-
speci fi c adaptations, comparisons with more related organisms are
much more informative. Until recently, such comparative studies
were hampered by the fact that beyond morphological descriptions
very little was known about any or just a few of the closer cousins of
D . discoideum . A number of advances are changing this state of
affairs. DNA- or protein sequence-based phylogenetic analyses have
clari fi ed relationships between the major divisions of eukaryotes and
groups within these divisions. Dictyostelia are now robustly placed
within Amoebozoa, a deeply rooted diverse group of mostly unicel-
lular organisms. Genome sequencing projects, particularly the recent
advent of high throughput genome sequencing, have revealed the
protein coding potential of protists that are representative of major
groups and divisions. Development of gene manipulation strategies
has made more protists amenable for studies into the molecular
mechanisms that control their physiology and life cycle transitions.
In this chapter, we fi rstly discuss the classi fi cation of Amoebozoa,
their position in the tree of life and the morphologies and life styles
that de fi ne the major groups. We next discuss roads to multicellu-
larity in all eukaryote divisions and fi nally zoom in on the
Dictyostelia and summarize recent insights into the evolution of
multicellularity in this group.
The morphology-based fi ve kingdom classi fi cation of all living
organisms (bacteria, protists, animals, plants, and fungi) has in the
past 20 years been thoroughly uprooted by molecular sequence
data. Instead, now three domains of life are recognized—eubacteria,
archaea, and eukaryotes— (
4 ) and eukaryotes are now partitioned
into six kingdoms or divisions— Excavata , Rhizaria , Chromalveolata ,
Plantae , Opisthokonta, and Amoebozoa (
3, 5, 6 ) . There is further-
more reasonable molecular and morphological support for a basic
dichotomy of the eukaryotes into two superclades, unikonts and
bikonts. Unikonts comprise the Amoebozoa and Opisthokonta , a
clade that contains the Metazoa, Fungi, and associated unicellular
relatives, while bikonts comprise the remaining divisions (Fig.
1a ).
Unikonts usually have only one cilium or fl agellum with an associ-
ated centriole, whereas bikonts ancestrally harbor two centrioles
and cilia. Bikonts undergo ciliary transformation by converting a
younger anterior cilium into a modi fi ed older posterior cilium.
Unikonts may have two or more cilia or fl agella, but in such cases
the anterior one never transforms into a posterior one. Several
groups within the bikonts have acquired photosynthetic endosym-
bionts or chloroplasts, but this is not the case for unikonts (
7– 9 ) .
2 Classi fi cation of Amoebozoa
2.1 Position in the
Tree of Life and
General Morphology

3Amoebozoa
However, the novel classi fi cations which are mostly based on
sequences of just one gene (small subunit ribosomal RNA) are still
in a state of fl ux, and phylogenies based on more molecular mark-
ers are badly needed.
Amoebozoa characteristically have no de fi ned shape and are
constantly changing form by extending protrusions known as pseu-
dopodia. However, this property is not unique to Amoebozoa or
even to unikonts; other amoeboid groups, such as the Heterolobosea
and Filosea, are actually members of the bikont divisions Excavata
and Rhizaria (
10, 11 ) . Many Amoebozoa alternate a unicellular
trophozoite stage with one or several different life cycle transitions.
The most common transition is the formation of a dormant cyst in
response to environmental stress. The protostelid amoebas can
additionally form a stalked spore. Sexual fusion of myxogastrid
amoebas followed by nuclear division results in a large syncytial cell
Fig. 1 Phylogenetic relationships between major eukaryote divisions and
Amoebozoa. ( a ) Schematic representation of the eukaryote tree of life. The
eukaryotes are currently subdivided into the six major divisions of Excavata,
Rhizaria, Chromalveolata, Plantae, Opisthokonta, and Amoebozoa, with the latter
two considered to form a larger unikont clade, while the remaining divisions
group together as bikonts ( 3, 5, 6 ) . ( b ) Relationships between major groups of
Amoebozoa. The current consensus phylogeny of Amoebozoa is based mainly on
SSU rRNA sequences and morphological features ( 18, 28, 31 ) . The positions of
the polyphyletic protostelids are indicated by arrows . Triangles indicate relative
species richness of groups, but are not exactly to scale

4 Christina Schilde and Pauline Schaap
that continues to feed and eventually forms spore-bearing structures
in response to environmental cues (
12 ) . Sexual fusion of Dictyostelid
amoebas followed by cannibalistic engulfment of other amoebas
leads to production of a dormant macrocyst (
13 ) , while colonial
assembly of amoebas to form spore-bearing structures is common
to both Dictyostelid and Copromyxid amoebas (
14 ) . Amoebas can
range in size from a tiny 3–5 μ m for the aptly named Parvamoeba
(
15 ) up to 5 mm for Pelomyxa palustris ( 16 ) . The stalks of the
Dictyostelid D . giganteum can reach over 7 cm (
17 ) , and the plas-
modia of Myxogastrids can cover areas of up to several square
meters, making them the largest unicellular organisms (
12 ) .
Amoebozoa can be further divided into the phyla Conosa , which
either have cilia or a fl agellum or have secondarily lost them; Lobosa ,
which never have cilia or fl agella; and the free-living, anaerobic,
fl agellated Breviatea (Fig.
1b ) ( 18, 19 ) . Breviata anathema is a
marine amoebo fl agellate with irregular, pointed, and sometimes
branched pseudopodia. Cells are sometimes multinucleate and
Breviata can form cysts. Although it lacks mitochondria, Breviata
contains nuclear mitochondrial genes, indicating that mitochondria
were lost during its adaptation to an anaerobic habitat (
19, 20 ) .
The phylum Lobosa can be divided into the well-supported
Tubulinea and the less well-de fi ned Discosea . Tubulinea have a
more or less cylindrical shape and show typical amoeboid movement
through pseudopod extension and cortical contraction. Tubulinea
comprise the naked amoeba genera Amoeba , Chaos , Copromyxa ,
Hartmanella , Leptomyxa , Gephyramoeba , and Echinamoeba and
the testate amoebas or Arcellinida (
5, 18 ) . Testate amoebas pos-
sess an outer shell with a single opening, which consists either
entirely of secreted proteins as in Arcella , a mixture of secreted and
captured organic material as in Dif fl ugia , or secreted anorganic
material as in Quadrulella . All testate amoebas are free living in soil
and freshwater and can encyst inside the shell under unfavorable
conditions. Within Discosea , amoebas with variable, often fl attened
shapes are combined, and they do not necessarily form a natural
group (
18, 21 ) . Typical examples of Discosea are Acanthamoeba ,
Vanella , Dermamoeba , and Thecamoeba . Cyst formation occurs in
Acanthamoeba and some other Discosea , but the life cycles of most
Discosea are unknown.
The phylum Conosa comprises the Variosea , Archamoebae , and
Mycetozoa (
18, 22 ) . Flagellated species of Conosa are characterized
by a cone of microtubules that connects the mostly single basal
body to the nucleus, forming a so-called karyomastigont. Variosea
contain dissimilar species, such as the fl agellate Phalansterium (
22 )
and the multiciliated amoeba Multicilia marina (
23 ) , in which,
respectively, the fl agellum or cilia each have an apposed basal body.
Phalansterium solitarium is a solitary species that can form cysts,
while Phalansterium digitatum forms colonies in which cells are
2.2 Phylogenetic
Relationships and
Specializations

5Amoebozoa
embedded in a globular organic matrix ( 22, 24 ) . The Varipodida
are also grouped with Variosea and contain species with thin and
sometimes branched pseudopods like Acramoeba , Grellamoeba ,
Filamoeba, and Flamella (
22 ) .
The Archamoebae , which have secondarily lost their mitochon-
dria (
25 ) , contain the anaerobic Mastigamoebida , comprising
Mastigamoeba , Endamoeba , and Endolimax , and the Pelobiontida ,
comprising Pelomyxa , Entamoeba , and Mastigina . In Mastigamoeba
the nucleus is physically attached to the basal body of a forward fac-
ing fl agellum and can be protracted. It can both encyst and form
multinucleate cells. Endamoeba and Endolimax are found in the
guts of animals and spread as cysts. Pelomyxa palustris is a multi-
nucleate amoeba, containing up to several thousand nuclei (
26 ) .
Pelomyxa lives in anaerobic freshwater sediments, where it indis-
criminately takes up material and digests usable constituents. Besides
inclusions like sand and diatom shells, the cytoplasm harbors several
bacterial endosymbionts, some of which are methanogen. Its life
cycle is complex with binucleate cells being derived by plasmotomy
from larger cells or hatched from cysts and cysts with four nuclei.
Most Entamoeba species are harmless commensals residing in the
large intestine of animals. Lacking mitochondria, they can only sur-
vive outside the body as dormant cysts. E . histolytica is an important
human pathogen in developing countries, which causes amoebic
dysentery and often lethal liver abscess (
27 ) .
The Mycetozoa , characterized by spore-bearing fruiting bodies,
are the most diverse group within the Amoebozoa and comprise
some protostelids, the Myxogastria and the Dictyostelia. The pro-
tostelids are however a larger polyphyletic assemblage and fall into
different lineages within the Amoebozoa (Fig.
1b ) ( 28 ) . Most pro-
tostelids form a single spore on top of a thin hollow stalk that is
secreted by the same cell, but species like Protosporangium (
29 ) can
have up to four spores. Many species also form cysts and in some
species, amoeba fuse to form small multinucleate plasmodia. The
Myxogastria group into two clades containing either the dark-
spored Physarida and Stemonitida or the bright-spored Liceida and
Trichiida (
30, 31 ) . Myxogastrids are amoebo fl agellates and hatch
from spores either as amoebas or bi fl agellated cells. These forms can
also interconvert, with wet conditions favoring the fl agellate form.
Upon nutrient depletion either cell type encysts to form a dormant
microcyst. In addition, both amoebas and fl agellates can fuse to
form a zygote when compatible mating types are present. The
zygote then goes through multiple synchronous nuclear divisions
without cytokinesis and continues to feed, thus causing large to
enormous single-celled plasmodia to form. Under dry and/or cold
conditions, the plasmodium converts into irregular hardened masses
of dormant macrocysts, called sclerotia. Other environmental stim-
uli, such as light, induce cleavage of the plasmodium into segments
with single nuclei that mature into haploid spores, after going

6 Christina Schilde and Pauline Schaap
through one round of meiosis. The remainder of the protoplasm
forms often quite intricate structures to elevate the spore mass
above the substratum (
12, 32 ) .
The Dictyostelia form multicellular fruiting bodies by aggrega-
tion of amoebas and are commonly found in forest soils. They have
been isolated from the Arctic to the tropics (
33 ) , but there are no
marine species and as yet only one description of a pathogenic
D . polycephalum isolate (
34 ) . The amoebas phagocytose bacteria
and small yeasts, although one species, D . caveatum , can also eat
other amoebas by nibbling (
35, 36 ) . Upon starvation, amoebas
secrete a chemoattractant, which can be cAMP, glorin, folate, a
pterin, or an as yet unidenti fi ed compound, and form an aggregate
consisting from around ten to a million cells (
1, 37 ) . Some species
form an intermediate pseudoplasmodium or “slug” that moves
towards warmth and light to fi nd a suitable spot for fruiting body
formation. Once aggregated, the amoebas initiate differentiation
into condensed encapsulated spores and highly vacuolated stalk
cells. Stalk cells are encased by a cellulose wall and are collectively
shaped into a rigid column by a cellulose stalk tube, which carries the
spore mass above the substratum (
38 ) . Ancillary structures, called
upper and lower cups, which support the spore mass, and a basal
disk to anchor the stalk to the substratum can also be present (
39 ) .
The spores are hydrophilic and are most likely dispersed by rain
and melting snow, but small soil invertebrates and even birds and
bats may also aid in spore dispersal (
40, 41 ) .
Many Dictyostelid species can also encapsulate individually as
microcysts or engage in sexual fusion and form macrocysts, a pro-
cess in which the zygote attracts and ingests other amoebas before
surrounding itself with a thick wall. After a long period of dor-
mancy, the macrocyst undergoes meiosis and multiple mitoses and
eventually hatches to yield several haploid amoebas (
13, 42 ) .
Population genetics of wild isolates indicates that mating occurs
frequently in nature (
43 ) . Speci fi c environmental conditions trigger
entry into the alternative pathways of fructi fi cation, microcyst-,
or macrocyst formation. Fructi fi cation requires an air-water interface
and is stimulated by light. Microcysts are formed under dark,
humid, or submerged conditions with high solute or ammonia levels
as additional stimuli (
44 ) . Macrocyst formation usually requires
the presence of a compatible mating type and is stimulated by eth-
ylene, darkness, and submersion (
45 ) .
Traditionally, the Dictyostelia have been divided into the gen-
era Acytostelium , with a secreted acellular stalk, Dictyostelium with
unbranched or irregularly branched sorocarps, and Polysphondylium
with regular whorls of side branches (
44 ) . However, molecular
phylogenetic analysis revealed that Dictyostelia can be subdivided
into four major groups, called groups 1–4, with Dictyostelids
being present in each group and multiple independent origins
for Polysphondylid-like species (
46 ) . Extension of taxon sampling

7Amoebozoa
indicated that a few group-intermediate species, such as P . violaceum ,
D . polycarpum , and D . polycephalum , may represent additional
minor clades (
47 ) .
In summary, the Amoebozoa are a division of amoeboid or
amoebo fl agellate organisms that most commonly alternate a tro-
phozoite feeding stage with a dormant cyst stage. Sexual fusion is
also common, leading either to formation of dormant zygotic cysts
or to multinucleate cells of varying sizes. Several subdivisions have
evolved forms that elevate one or a few spores above the substra-
tum, but only Copromyxa and Dictyostelia construct fruiting struc-
tures from more than ten cells.
Multicellularity arose several times independently during evolution
but is commonly perceived to be only present in plants, animals,
and fungi. However, most eukaryotic divisions and phyla show
independent inventions of multicellularity. In the bikonts, multi-
cellular photosynthetic organisms evolved independently from uni-
cellular green algae (all land and many marine plants), brown algae
(kelps and stramenopiles), and red algae (many seaweeds). Not
only Dictyostelia and Copromyxa in Amoebozoa but also unrelated
amoebas like Acrasis and Pocheina in Excavata (
48 ) and Fonticula
alba in Opisthokonta (
49 ) form fruiting bodies from hundreds to
up to a million cells. The Opisthokonta on the unikont side is par-
ticularly prone to multicellularity with colonial forms in the
choano fl agellates (
50 ) , both unicellular and multicellular species in
Fungi and unconditional multicellularity in Metazoa . Both Metazoa
and vascular green plants generate the multicellular form from a
zygote through cell division and cell differentiation. In Fungi ,
mycelia of interconnected cells can develop by cell division from
either a zygote, a spore, or asexual propagates. On the other hand,
aggregative multicellularity does not necessarily depend on cell
divisions and almost always results in formation of a spore- or cyst-
bearing structure. In the following paragraphs, we describe organ-
isms with aggregative multicellularity in more detail.
The Chromalveolate ciliate species Sorogena stoianovitchae feeds
on the smaller ciliate Colpoda . When starved at high cell density,
Sorogena ciliates aggregate by cell adhesion to form a mound
encased in a mucous sheath (Fig.
2a ). This sheath then contracts
and elongates to form an acellular stalk that lifts the cell mass above
the water surface, followed by encystation of the ciliate cells (
51, 52 ) .
Some heterolobose amoeba genera in the Excavates , such as Acrasis
spp . and the related Pocheina rosea , either encyst individually or
amoebas aggregate to form a mound that is lifted above the sub-
stratum by virtue of cells encysting at the base of the structure.
Acrasis fruiting bodies are mostly tree-shaped with the mature
spores forming branched chains (Fig.
2b ), while Pocheina forms a
stalk with a globose spore mass. Both stalk and spore cells are viable
and only marginally differ from each other. They are also very similar
2.3 Many Roads
to Multicellularity

8 Christina Schilde and Pauline Schaap
Fig. 2 Fruiting body formation in various organisms with aggregative multicellu-
larity. ( a ) The ciliate S . stoianovitchiae aggregates by adhesion and forms a
sheath that contracts to form a stalk, while the cells encyst. ( b ) Acrasis amoebas
aggregate and form a stalk by encysting at the base of the structure, while more
apical cells rearrange themselves into chains and then encyst. ( c ) F . alba amoe-
bas aggregate and deposit a cone-shaped matrix around the cell mass. Amoebas
differentiate into spores and are expulsed through the apex. ( d ) Copromyxa
amoebas are attracted to a few encysted founder cells. Once aggregated, cells
crawl on top of existing cysts and then encyst themselves

9Amoebozoa
to cysts but additionally have plinth-like connecting structures,
called hila (
5, 48, 53, 54 ) .
Amoebas of the amoeboid Opisthokont Fonticula alba collect
into aggregates and secrete an extracellular matrix that forms a
volcano-shaped enclosure around the cells (Fig.
2c ). When the
amoebas mature into spores, the apex opens eruptively and depos-
its the spores as a droplet on top of the structure, leaving some
undifferentiated amoebas behind at the base (
49, 55 ) . The
Amoebozoan Copromyxa protea feeds on bacteria in dung and,
similar to Dictyostelia, can enter upon three alternative survival
strategies when starved. Amoebas can differentiate into round or
“puzzle-piece”-shaped (micro)cysts or fusion of two amoebas
results in the formation of dormant double-walled spherocysts.
Alternatively, some amoebas encyst fi rst and then become founder
cells, which attract other amoebas to form an aggregate (Fig.
2d ).
The amoeba in the aggregate form a branched fruiting structure by
crawling on top of each other and in turn forming so-called soro-
cysts that are morphologically identical to microcysts (
14, 56 )
Evidently, the formation of fruiting bodies by aggregation has
evolved several times independently. Despite occurring in very
diverse genetic lineages, these forms of multicellularity all resemble
Dictyostelia in the fact that they are a response to starvation and
generate a structure that elevates dormant spores or cysts above an
air/water interface. Most aggregating amoebas or ciliates use
fructi fi cation as an alternative strategy to encystation of individual
cells in situ. However, Dictyostelia are unlike all aggregating amoe-
bas by differentiating into at least two morphologically distinct cell
types and by the sophistication of their aggregation process and
morphogenetic program.
Outwith Dictyostelia, aggregating species usually collect into
mounds by cell adhesion or by moving towards each other indi-
vidually. While the latter mode is also observed for some of the
smaller species of Dictyostelia, most Dictyostelids aggregate as
interconnecting streams of amoebas. Studies in the model organ-
ism D . discoideum revealed that this mode of aggregation results
from relay of chemoattractant waves, in this case cAMP, through
the starving population (
57 ) . A biochemical network, consisting
of the cAMP receptor, cAR1, the adenylate cyclase, ACA, the
extracellular phosphodiesterase PdsA, and intracellular proteins,
including PKA and RegA, generates pulses of cAMP in a few
starving cells (
58, 59 ) . In surrounding cells these pulses elicit
cAMP-induced cAMP secretion (cAMP relay), which results in
propagation of the cAMP pulse throughout the cell population
and chemotactic movement of cells towards the cAMP source.
Once aggregated, the tips of multicellular structures continue to
emit cAMP pulses, which guide and shape the organism during
slug migration and fruiting body formation by coordinating the
2.4 Evolution
of Morphogenesis
in Dictyostelia

10 Christina Schilde and Pauline Schaap
movement of its component cells ( 60 ) . Secreted cAMP not only
coordinates morphogenesis but also regulates stage- and cell-
type-speci fi c gene expression. Nanomolar cAMP pulses accelerate
the expression of aggregation genes (
61 ) , while micromolar
cAMP concentrations induce the expression of prespore genes
and inhibit stalk gene expression (
62, 63 ) .
In addition to these roles for secreted cAMP, intracellular
cAMP acting on PKA also crucially regulates many developmental
transitions. Together with secreted cAMP, intracellular cAMP is
required for prespore differentiation (
64 ) . Furthermore, active
PKA crucially triggers spore and stalk maturation and maintenance
of spore dormancy in the fruiting body (
64– 66 ) . For stalk and
spore maturation, cAMP is produced by adenylate cyclase R (ACR)
and for induction of prespore differentiation and control of spore
germination by adenylate cyclase G (ACG) (
66– 68 ) . ACG harbors
an intramolecular osmosensor and is activated by high ambient
osmolarity, a condition that keeps spores dormant in the spore
head (
69, 70 ) . The cAMP phosphodiesterase RegA also plays a
crucial role in regulating intracellular cAMP levels (
71 ) .
Recent comparative studies into conservation and change in
genes involved in synthesis and detection of cAMP throughout the
Dictyostelid phylogeny provided insight into the evolutionary origin
of cAMP signaling. Osmolyte-activated ACG is functionally con-
served throughout the Dictyostelid phylogeny (
72 ) . Many early
diverging Dictyostelid species have retained the ancestral mechanism
of encystation (
46 ) . Similar to spore germination, cyst germination is
also inhibited by high osmolarity, but unlike spore formation, encys-
tation can be directly induced by high osmolarity. For soil amoebas,
high osmolarity is probably a signal of approaching drought, which
increases the concentration of soil minerals. Osmolyte-induced encys-
tation is mediated by cAMP production and PKA activation (
72 ) ,
suggesting that the roles of intracellular cAMP and PKA in spore dif-
ferentiation and germination are evolutionary derived from a similar
role in the encystation of solitary amoebas.
Genes encoding cAR1 and therefore extracellular cAMP sig-
naling are also functionally conserved throughout the Dictyostelid
phylogeny. In group 4 species, such as D . discoideum and
D . rosarium , cAR1 is expressed from a proximal promoter during
postaggregative development and from a distal promoter during
aggregation (
73, 74 ) . In these species, inhibition of cAR function
blocks both aggregation and subsequent development. Remarkably,
in groups 1 and 2, cAR1 orthologs are only expressed after aggre-
gation (
74 ) , and in either group 1, 2, or 3, abrogation of cAR
function disrupts slug and fruiting body formation, but not aggre-
gation (
75 ) . The latter effect was not unexpected, since group 1–3
species use other attractants than cAMP to aggregate, with glorin
being most prevalent (
76 ) . However, the fact that postaggregative
morphogenesis is blocked by loss of cAR function suggests that all

11Amoebozoa
Dictyostelia use oscillatory cAMP signaling to coordinate cell
movement during slug and fruiting body formation. This hypoth-
esis was further substantiated by recent observations that loss of
the PdsA gene from a group 2 species also resulted in disruption of
postaggregative morphogenesis, while aggregation remained nor-
mal. Strikingly, the af fi nity of PdsA for cAMP in groups 1–3 was
low but increased 200-fold in group 4 species. This probably
re fl ects an adaptation from hydrolyzing relatively high extracellular
cAMP concentrations within an aggregate to hydrolyzing much
lower concentrations in a dispersed fi eld of starving cells (
77 ) .
Taken together, the data indicate that oscillatory cAMP signaling
evolved fi rst to coordinate morphogenesis and that its additional
role in mediating aggregation appeared more recently in group 4.
cAR gene disruption in the group 2 species P . pallidum yielded
stunted fruiting structures that contained cysts instead of spores in
the spore head. This was due to the fact that the cAR null mutant
no longer expressed prespore genes in response to cAMP stimula-
tion (
75 ) . As discussed above, sporulation and encystation both
require intracellular cAMP acting on PKA, but sporulation addi-
tionally requires extracellular cAMP acting on cARs. With the lat-
ter pathway ablated, the cAR null cells reverted to the ancestral
strategy of encystation.
Together, these results suggest that cAMP signaling in Dictyostelia
evolved from a “classical” second messenger role for cAMP in stress-
induced encystation (Fig.
3 ). Dictyostelia secrete most of the cAMP
that they produce but can only accumulate the micromolar concen-
trations that are required for prespore differentiation, once they have
aggregated. In early Dictyostelids, accumulation of secreted cAMP
Fig. 3 The evolution of morphogenetic cAMP signaling in Dictyostelia. Putative scenario for the evolution
of developmental cAMP signaling in Dictyostelia from a second messenger function in Amoebozoan encystation.
LCA last common ancestor

12 Christina Schilde and Pauline Schaap
may therefore have acted as a signal for the aggregated state and
have prompted cells to form spores and not cysts. Oscillatory cAMP
secretion, which requires cAR-mediated positive and negative feed-
backs on cAMP synthesis by ACA evolved next and enabled the cells
to form architecturally sophisticated fruiting bodies. cAMP-mediated
aggregation was the most recent innovation and only occurred in
group 4 (Fig.
3 ).
While at fi rst sight the multitudinous roles of cAMP in
D . discoideum in aggregation, morphogenesis, and gene regulation
may seem perplexing, evolutionary reconstruction of these roles
allows us to separate core ancestral processes from more recent
adaptations. In essence, evolutionary reconstruction reveals the
underlying logic of convoluted interrelated processes. Comparative
analysis and evolutionary reconstruction are therefore powerful
tools to unravel complex biological processes.
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