Apoptosis (2010) 15:269–278
DOI 10.1007/s10495-009-0442-7
UNUSUAL MODEL SYSTEMS FOR CELL DEATH RESEARCH
Apoptosis in pre-Bilaterians: Hydra as a model
Margherita Lasi • Charles N. David •
Angelika Böttger
Published online: 30 December 2009
Ó Springer Science+Business Media, LLC 2009
Abstract Hydra is a member of the ancient metazoan
phylum Cnidaria and is an especially well investigated
model organism for questions of the evolutionary origin of
metazoan processes. Apoptosis in Hydra is important for the
regulation of cellular homeostasis under different conditions
of nutrient supply. The molecular mechanisms leading to
apoptosis in Hydra are surprisingly extensive and compa-
rable to those in mammals. Genome wide sequence analysis
has revealed the presence of large caspase and Bcl-2 fami-
lies, the apoptotic protease activating factor (APAF-1),
inhibitors of apoptotic proteases (IAPs) and components of a
putative death receptor pathway. Regulation of apoptosis in
Hydra may involve BH-3 only proteins and survival path-
ways, possibly including insulin signalling.
Keywords Apoptosis
Hydra
Evolution
BH-3 only

Bcl-2
Caspase
Introduction
Cnidaria, Porifera, Ctenophora and Placozoa are the four
ancient phyla that diverged from the metazoan lineage
before the evolution of Bilaterians. Studying model
organisms from these phyla is expected to shed light on the
evolution of multicellularity in the animal kingdom. Thus,
a number of new model organisms have emerged from the
M. Lasi
C. N. David
A. Böttger (&)
Department Biology II, Biozentrum, Ludwig-Maximilians-
University Munich, Großhaderner Str. 2, 82152 Planegg-
Martinsried, Germany
e-mail: boettger@zi.biologie.uni-muenchen.de
C. N. David
e-mail: david@zi.biologie.uni-muenchen.de
pre-bilaterian metazoan phyla in recent years. EST and
whole genome sequencing projects for these models have
revealed an intriguingly high complexity of molecular
developmental pathways in early metazoans.
The transition from single-celled to multi-cellular
organisms required cooperation between different somatic
cell types. Cells had to divide tasks requiring some to
proliferate, others to differentiate and, as we know now, a
number of them to die. Forms of programmed cell death
are found in all organisms including the slime mold
Dictyostelium, protozoans (e.g. Tetrahymena), algae (e.g.
Volvox), fungi (Saccharomyces) and plants (reviewed in
[1]). The term apoptosis is usually used for those forms
of programmed cell death that are based on the action of
caspases and Bcl-2 proteins and involve phagocytosis of
dying cells. These appear to have evolved only in the
animal lineage. It is therefore informative to look at model
organisms from ancient animal phyla to understand how
apoptotic pathways may have evolved.
The cnidarian model organism Hydra
The fresh water polyp Hydra clearly is the most thoroughly
studied non-bilaterian animal. The first experiments on the
Cnidarian Hydra were done in the mid eighteenth century
immediately after the polyps had been discovered. They
revealed an astonishing capacity of these animals to
regenerate which extends to complete regeneration of
polyps from dissociated cells [2].
Hydra has a simple body plan (Fig. 1). It has one body
axis with hypostome and tentacles at the apical end and a
peduncle and basal disc at the basal end. It develops from
the two germ layers after gastrulation of the embryo. The
endodermal layer develops into the inner gastrodermis and
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270 Apoptosis (2010) 15:269–278

a junctions including adhering belt desmosomes, septate

tentacle junctions and gap junctions [6–8]. Epithelial cells in the
Hydra body column proliferate continuously and are dis-
placed towards the apical end into tentacles and towards
hypostome the basal end into the peduncle and basal disk and into buds
(Fig. 1a). Cells in the basal disk and in the tentacles stop
body column
proliferating and differentiate into specialised cell types,
e.g. battery cells in the tentacles and foot cells in the
peduncle. They are continuously sloughed off at the ends of
bud peduncle
the animals and replaced by newly arriving cells from the
basal disk proliferation zone in the body column. Cells at the apical
tip of the animal constitute a second proliferation zone and
differentiate into hypostome cells. Thus, both epithelia are
b constantly renewed [9–11].
In addition to the two epithelial cell layers Hydra has
specialised cells. These include nerve cells, which form a
nerve net extending throughout the entire animal but not
forming a brain or central ganglia. Gland cells are mostly
nucleus present between the epithelial cells of the gastrodermis
apo where they secrete digestive enzymes into the gastric
cavity. The name-giving cell type for the phylum cnidaria
are nematocytes, also called cnidocytes. These cells con-
tain a nematocyst capsule, which is used to capture prey.
They are incorporated into battery cells of the tentacles
endodermal epithelial cell with apoptotic where they remain until used for stinging and poisoning of
cell in phagocytic vacuole
prey animals. Finally, Hydra produces male and female
gametes from germ cells for sexual reproduction. All these
c specialised cell types are products of the pluripotent
interstitial stem cell lineage. Interstitial cells are located in
the spaces between the epithelial cells, mostly in the epi-
dermis. They constitute a self-renewing proliferating stem
cell population, part of which differentiates in each cell
nucleus generation leading to the formation of the above mentioned
cell types [9, 12–14].
apo

Apoptosis in Hydra

ectodermal epithelial cell with two apoptotic
cells in phagocytic vacuoles
Fig. 1 a Schematic drawing of an adult Hydra with bud. Hypostome,
tentacles, body column, peduncle and basal disk are indicated; b and c
ectodermal and endodermal epithelial cell from macerates of Hydra
cells; left hand panels show phase contrast; right hand panels show
DNA stain with DAPI. In these images nuclei are stained as indicated
by arrows. Apoptotic cells (apo) in phagocytic vacuoles show
pycnotic chromatin (arrows). Some food granules in the endodermal
cell are also stained with DAPI
the ectodermal layer into the outer epidermis. Epidermis
and gastrodermis are separated by the acellular mesoglea,
an extracellular matrix containing laminin and collagen
[3–5]. Epidermis and gastrodermis are polarised and sealed
epithelia containing cells that are joined by intercellular
The permanent self-renewal of all Hydra tissues implies
the need for tight regulation of cellular homeostasis,
especially under conditions of changing growth rate. Hydra
responds to limiting food supply by slowing down growth.
The budding rate decreases and the animals become
smaller [15]. However, corresponding changes in the epi-
thelial cell cycle are not observed [15, 16] i.e. cell pro-
duction continues although cell numbers do not increase. In
this situation a large number of newly produced cells are
phagocytosed by neighbouring cells [16]. This was first
shown in Feulgen-DNA stained preparations of starving
animals where significant numbers of pycnotic nuclei were
found in round phagocytic vacuoles mainly in gastrodermal
epithelial cells. Later re-examination of the cells in phag-
ocytic vacuoles clearly indicated that they were apoptotic.

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Apoptosis (2010) 15:269–278
They showed the typical apoptotic DNA distribution when
stained with DAPI (Fig. 1 b,c) and were TUNEL-positive
[17, 18]. Colchicine treatment, which disrupts the cell
cycle, induces massive cell death in Hydra and the dying
cells are phagocytosed by epithelial cells of the gastro-
dermis [17]. The degradation of DNA in colchicine treated
animals was demonstrated later to result in DNA laddering,
confirming that cell death in Hydra is apoptotic [17].
Furthermore, apoptotic vacuoles could be stained with
acridine orange (AO) [17, 18] due to the fact that AO
accumulates at the acidic pH in the vacuoles, as shown
before in Tetrahymena and in Drosophila [19, 20].
Regulation of cell numbers is an important physiological
function of apoptosis in Hydra. However, there are several
other situations where apoptosis has been shown to occur
in Hydra including interspecies grafts and regeneration
[21–26] and in response to cytostatic agents and irradiation
[27–29].
A very intriguing special case of apoptosis is observed
during differentiation of germ cells in Hydra. This applies
to both, the male and the female germ line [30–32]. Here we
will only discuss oogenesis. Germ cells differentiate from
the interstitial stem cell lineage. As a first step in oogenesis
a population of interstitial cells proliferates and forms a
so-called egg-patch at the side of the mother animal. Cells
in this egg patch later exit the mitotic cycle and start meiotic
S-phase. Their appearance changes, they accumulate gran-
ules in their cytoplasm and they enlarge. A few of these
cells then enter meiotic diplotene and grow even further.
The others start to transfer their cytoplasm into these dip-
lotene cells. At some point the few developing oocytes fuse,
so that only one oocyte remains, continually increasing in
Table 1 Comparison of apoptotic proteins present in Hydra, Caenorhabditis, Drosophila and mammals
Apoptotic proteins in different model organisms
Hydra Caenorhabditis
Caspases HyCaspA, B, C, D, E, F, Ced 3
G, H, I, L, M Csp-1, Csp-2
HyCARD 1, 2
HyDEDCasp
HyDDCasp
APAF-1 HyAPAF-1 Ced-4
Bcl-2 proteins: HyBcl-2-like 1, 2, 3, 4, 5, Ced-9
multi-domain 6, 7 HyBak-like 1, 2
Bcl-2 proteins: HyBH3-only 1, 2, 3, 4 Egl-1
BH3-only
IAP HyIAP – DIAP1, 2
Death receptor HyTNFR-like receptor – Wengen
FADD HyFADD – dFADD
BH3 only proteins of the BNip family are present in all three organisms and have been omitted from the table
271
size through cytoplasm transfer from all the other germ
cells. The latter cells shrink and finally, their remains are
phagocytosed by the oocyte. The morphological features of
their nuclei suggest apoptosis. They are positive with
TUNEL and their chromatin appears pycnotic [31]. The
fascinating thing is that they are not further degraded after
phagocytosis by the oocyte. The oocyte instead keeps them
enclosed in phagocytic vacuoles even after fertilisation.
During embryonic development they are distributed to the
endodermal cells of the embryo and only get degraded after
the new polyp has hatched [32]. How this arrest in apoptosis
is achieved is not known at present.
Molecular mechanisms for apoptosis are conserved
in all animals
Molecular mechanisms of cell death are conserved
throughout animal evolution. They involve caspases, Bcl-2
proteins, adaptor proteins, death receptors and inhibitors of
apoptotic proteases (IAPs) (see Table 1).
Caspases are proteases with active-site cysteine resi-
dues. Their activation from inactive pro-caspases is the
critical step in apoptosis and occurs through an amplifying
cascade from upstream initiator caspases to downstream
executioner caspases. The autocatalytic activation of initi-
ator caspases requires a local increase in their concentra-
tion. This is achieved by adaptor proteins, which recruit
pro-caspases through homotypic protein–protein interac-
tions via DED-domains or CARD-domains. These domains
are present in both the caspase pro-domains and the adaptor
proteins [33, 34].
Drosophila Homo
Dredd, Dronc, Dream, Caspase 1, 2, 3, 4, 5, 6, 7,
Dcp-1, DRICE, Decay, Daydream 8, 9, 10, 12, 13, 14
DARK APAF-1
Debcl, Buffy Bcl-2, Bcl-xL, Bcl-W, Mcl-1,
Bcl-B, Bcl-2A1, Bax, Bak, Bok
– Bid, Bmf, NOXA, PUMA, Bad,
Bim, Bik, Hrk
XIAP, NAIP, c-IAP1, 2
TNF-R 1,2 CD95, CD 40,
p75 and many others
FADD
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Extrinsic apoptosis induction requires death receptors
represented by homologs of the TNF-receptor family.
Binding of ligands such as TNFa or FasL leads to receptor
trimerisation and clustering. The adaptor proteins TRADD
and/or FADD and pro-caspases with DED domains (e.g.
pro-caspase 8) are recruited to these clusters and interact
via DED-domains forming a so-called death inducing sig-
nalling complex (DISC) at the membrane. This leads to
autoactivation of pro-caspases with DED domains and
subsequent activation of executioner caspases [35, 36].
Intrinsic apoptosis induction is mediated by the apop-
tosome, a protein complex including APAF-1 and pro-
caspases with CARD-domains. In mammals formation of
the apoptosome requires cytochrome C, which is released
from mitochondria. Mitochondrial integrity in turn is reg-
ulated by the Bcl-2 protein family with its multi and single
domain members exhibiting pro- and anti-apoptotic func-
tions. Anti-apoptotic members of this family are integrated
into the outer mitochondrial membrane and inhibit pro-
apoptotic family members. Apoptosis is induced by single
domain (or BH3 only) Bcl-2 family members, which
respond to stimuli such as DNA damage or the absence of
survival signals. This changes the equilibrium between the
multi-domain pro- and anti-apoptotic Bcl-2 proteins and
leads to mitochondrial membrane permeabilisation and
cytochrome C release [37].
Further components of apoptotic pathways are IAPs,
inhibitors of apoptotic proteases. They contain character-
istic zinc-finger motifs (BIR-domains: baculoviral IAP
repeats) and directly inhibit the catalytic activity of casp-
ases. In addition, many IAPs have a RING-domain and thus
E3 ubiquitin ligase activity and mediate the proteasomal
degradation of caspases (reviewed in [38]).
Caspases and Bcl-2 family proteins have been found in
sponges, cnidarians and all bilaterian clades [39–41]. Their
function in apoptosis has been best studied in mammals
and in the major invertebrate model organisms Caeno-
rhabditis elegans and Drosophila melanogaster [42].
C. elegans has only one member of the Bcl-2 family
(ced-9), one caspase, which has a CARD-domain and is
involved in apoptosis (ced-3), the APAF-1 homolog Ced-4
and one BH3-only protein (egl-1), which initiates somatic
cell death. Cytochrome C can not bind to Ced-4 and thus
cytochrome C release from mitochondria is probably not
necessary for apoptosis in Caenorhabditis. Death receptors
are not known to participate in apoptosis in C. elegans,
and IAPs are not found encoded in the genome [43]
(with the exception of survivin, whose primary function in
C. elegans is not in apoptosis [44]).
Drosophila has seven caspases, five are effector casp-
ases, one has a CARD-domain and one has a DED-domain,
suggesting a more complex network for apoptosis in
comparison with Caenorhabditis. Two Bcl-2 family
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Apoptosis (2010) 15:269–278
members, Debcl and Buffy are also present in Drosophila,
although their precise function in apoptotic pathways is
not clear. Genetic studies have shown, that apoptosis in
Drosophila is primarily regulated via IAPs. Death stimuli
are relayed through expression of inhibitors of IAPs,
namely RPR, GRIM and HID [45]. A death receptor has
been described in Drosophila, but its activation does not
lead to caspase recruitment (it lacks the DD domain, which
is required for binding to FADD; [46–48]).
Mammals, by comparison with Caenorhabditis and
Drosophila, exhibit highly complex extrinsic and intrinsic
pathways for apoptosis induction [49]. They have a large
caspase family with 15 members, which are divided into
several groups including initiator and executioner caspases
(reviewed in [50]). Mammals also have an extended Bcl-2
family with pro and antiapoptotic members ([39] and
reviewed in [49]). In addition a large number of BH3-only
proteins is known, only one of which, BNip, is conserved
through evolution. Finally, mammals have four IAP genes
involved in apoptosis regulation.
This brief summary indicates that caspases and Bcl-2
proteins first evolved in porifera and that these gene fami-
lies increased in complexity from worms to mammals.
However, recent analyses of whole genome sequences from
cnidarians suggest that the caspase and Bcl-2 families were
already highly complex in cnidarians and that Caenorhab-
ditis and Drosophila lost many of the genes involved in
apoptosis. Ten genes encoding putative caspases, 11 genes
encoding putative Bcl-2 family members and several genes
encoding APAF-1 homologs and IAPs have been found
in genome searches of the sea anemones Aiptasia and
Nematostella [51, 52]. An even larger number of caspases
and Bcl-2 family proteins, including BH3-only proteins was
found in the hydrozoan Hydra magnipapillata. As described
above, apoptosis in Hydra has been investigated for many
years and now, with the complete set of apoptosis genes
available, studies elucidating their function are beginning.
The first results are reviewed in the following.
Hydra caspases and potential caspase activation
mechanisms
Lysates from Hydra in which massive apoptosis has been
induced by colchicine treatment exhibit a caspase 3 specific
enzymatic activity detectable with fluorogenic substrates
[17]. Genome and EST-library searches have now revealed
19 caspase genes. Two are incompletely covered by ESTs,
these have been omitted from the following analysis. Eight
have been cloned from cDNA (manuscript in preparation;
for HyCARDCasp1 Lange and Bosch, personal commu-
nication). Nine more are completely represented in the
EST-library (Fig. 2a, b). Analysis of these 17 sequences
Apoptosis (2010) 15:269–278 273

a b
Group 1
44 THGE QACQG GSWFM
44 HyCaspE
62
THGK QACQG GSWYI
HyCaspG

87 THGK QACRG GSWFV

HyCaspB
90
SHGE QACRG GSWFI
HyCaspC
THGE QACRG GSKFI
100
HyCaspF
SHGL QACQG GSWFI
HyCaspH
100 100
99 SHGN QACRG GSWFI
HyCaspD
SHGS QACRG GSWFI
HyCaspA
73
100 CARD SHGV QACRV GSYFI
HyCARD1
CARD SHGQ QACQG GSWFI
100 HyCARD2
SYGE ESCLG GSWFI
HyPseudocasp1
SIGD QTCTG NSWFI
100 100 HyPseudocasp3

100
Group 2
SHGF QACQG GSWYI
HyCaspI
DED SHGY QACQT GSWYI
HyDEDCasp
SHGF QACQG GSYII
HyCaspL

Group 3
DD AHGQ ESCRG SPWVI
HyDDCasp
AHGE DSCRG SPWVK
HyCaspM

DD DED CARD CARD

100 amino acids

Group3 Group2 Group1 scale bar

Fig. 2 a Phylogenetic tree on the basis of a ClustalW alignment of
caspase sequences. All sequences are represented by gene models
(hmas) in the Hydra genome (http://hydrazome.metazome.net) and
are covered by EST-sequences. Boxed caspase sequences have been
cloned from Hydra cDNA. TreeTop-graphic; (http://www.genebee.
msu.su/services/phtree_reduced.html); bootstraps are indicated;
branches with bootstraps \50 have dotted lines. Caspases are in 3
groups as indicated: group 1 contains two caspases with CARD
domains, group 2 contains one caspase with a DED domain and group
3 one caspase with a DD domain; b Schematic representation of
Hydra caspase sequences in groups 1, 2 and 3. Sequences divided into
prodomains, large subunits and small subunits are shown as bars
according to scale; sequences surrounding active site histidine and
cysteine residues and conserved substrate binding site in the small
subunits are indicated; CARD-, DED-and DD-prodomain signatures
are also shown where present. Accession numbers: Hydra caspase 3B:
AF155128; Hydra caspase 3A: AF155127; Hydra caspase 3C:
AY924233; Hydra caspase D: GU128636; Hydra caspase DD:
GU128637; Hydra CARD caspase 1: GU128638; Hydra CARD cas-
pase 2: GU128639; Hydra DED caspase: GU208869

shows that 15 have conserved active site residues, includ-
ing a cysteine and a histidine residue. Moreover, the sub-
strate pocket in the small subunit is also conserved in these
sequences as GSWF/Y. In addition aspartate residues rep-
resenting potential caspase cleavage sites between the large
and small subunits are conserved (Fig. 2b). Two of the 17
sequences with high homology to caspases lack the
important active site histidine residue. We thus call them
pseudocaspases (Fig. 2b).
Phylogenetic sequence comparison of all Hydra casp-
ases reveals that they fall into three groups (Fig. 2a). The
largest group contains two caspases with CARD-domains
and two pseudocaspases. All the other caspases in this
group have prodomains of varying length. These prodo-
mains, however, do not show signatures for CARD
domains although we have demonstrated experimentally
that HyCaspA is capable of autoprocessing in contrast to
HyCaspB, C and D, which are not (manuscript in prepa-
ration). The second group contains 3 caspases, one of
which has a DED domain and could thus represent a Hydra
caspase 8 homolog (although mammalian caspase 8
homologs have two DED-domains). The other members in
the group have prodomains with no recognisable signature.
The third group is represented by two unusual sequences.

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274 Apoptosis (2010) 15:269–278

HyDDCasp has a DD domain in its prodomain and is to our
knowledge the only caspase with such a prodomain.
The presence of potential initiator caspases with CARD,
DED and DD domains in Hydra suggests similar caspase
activation mechanisms to those known from mammals. This
includes both extrinsic and intrinsic caspase activation
mechanisms. Based on the occurrence of additional proteins
involved in both pathways it appears that both could be
operating in Hydra. For the intrinsic pathway, in addition to
two CARD-domain caspases, Hydra has an APAF-1
homolog shown schematically in Fig. 3a [53]. APAF-1 is a
central component of the human apoptosome. It has an
N-terminal CARD-domain for recruitment of CARD-
domains from initiator caspases, e.g.caspase 9. C-terminally
it has a wheel-like structure with two propellers, one formed
by the first seven WD40 repeats and the other by the
remaining six WD40 repeats [54, 55]. Both propellers are
thought to interact with cytochrome C. HyAPAF-1 also
has an N-terminal CARD domain that could potentially
interact with the Hydra CARD-domain caspases. In Hydra
APAF-1 only seven WD40 repeats are present (see Fig. 3a).
These could potentially form a wheel, but only one pro-
peller. It is therefore hard to predict whether the Hydra
protein would bind to cytochrome C. Experiments are
underway to show whether HyAPAF-1 is involved in cas-
pase activation and if cytochrome C plays a role in this.
For the extrinsic pathway in Hydra, in addition to a
DED-domain containing caspase, a FADD-like adaptor
protein and a potential death receptor have been identified.
The receptor has been cloned from Hydra cDNA
(Fig. 3b,c). Whether these proteins engage in a stimulus
induced death activation pathway remains to be analysed.
Moreover, it is tempting to speculate that the unusual
HyDDCasp could participate directly in the extrinsic
apoptosis induction pathway by interacting with the
DD-domain of the Hydra death receptor.

Fig. 3 a Schematic drawing of a APAF-1

the domain structure of the
Hydra APAF-1 (accession WD40 repeats
number: GU121227) homolog CARD NOD
in comparison with human
APAF-1 and C. elegans CED-4; HsAPAF-1
CARD domains, NOD-domains
and WD40-repeats are
indicated; single WD40
domains as black boxes; CeCed-4
b schematic drawing of the
domains of the Hydra TNF-
receptor like protein (accession
number: GU121226) sequence HyAPAF-1
as deduced from cloned cDNA;
TNF-R1 and R2 and
transmembrane (TM) domains b TNF-R
are indicated; c domain
structure of the Hydra FADD
homolog as deduced from the TNF-R1 TNF-R2 TM DD
gene model hma1.131332
(http://hydrazome.metazome. HyTNF-R-like
net); DD and DED-domains are
indicated; d domain structure of
Hydra IAP (accession number:
GU121225). Sequence was
c FADD
deduced from cloned cDNA; 3
BIR-domains and RING-
domain are shown in compari- DED DD
son with human XIAP HyFADD

d IAP

BIR1 BIR2 BIR3 RING

HsXIAP

HyIAP

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Apoptosis (2010) 15:269–278
Caspase activity in Hydra could potentially also be
regulated by IAPs or by pseudocaspases. One bona fide
IAP gene was cloned from Hydra cDNA. It encodes a
protein with 3 BIR domains and a C-terminal RING
domain (Fig. 3d, manuscript in preparation). Moreover, the
pseudocaspases in our first caspase group (Fig. 2a) could
potentially act as inhibitors of ‘‘real’’ caspases by com-
peting for substrate binding. Recent work in C. elegans has
hinted at such a possibility. The C. elegans caspase like
protein Csp-3 does not contain an active-site cysteine [56].
Loss of the csp-3 gene, however, caused increased cell
death. Moreover, Csp-3 interacted with Ced-3 and inhib-
ited its autoactivation (but not the activity of the processed
caspase 3) [43].
Hydra Bcl-2 protein family
The second large gene family we identified in the Hydra
genome are the bcl-2 genes. Nine such genes were found
and cloned from cDNA (Fig. 4). Protein sequence con-
servation is very strong in the BH1 and BH2 domains but
not in the BH3 and BH4 domains ([53] and manuscript in
preparation). Similar observations have been made by
BH4 BH3 BH1 BH2 TM
HyBcl-2-like 1
HyBcl-2-like 2
HyBcl-2-like 3
HyBcl-2-like 4
HyBcl-2-like 5
HyBcl-2-like 6
HyBcl-2-like 7
HyBak-like 1
HyBak-like 2
Fig. 4 Schematic drawing of conserved BH domains of the Hydra
Bcl-2 family proteins as deduced from cloned cDNAs. Dark grey
coding region, small squared boxes BH4 domains, longitudinal
stripes BH3 domains, diagonal stripes BH1 domain, dotted boxes
BH2 domains, black box membrane anchoring domain. Accession
numbers: Hydra Bak-like protein 1: EF104645; Hydra Bak-like
protein 2: EU035760; Hydra Bcl-2-like 1: EF104646; Hydra Bcl-2-
like 2: EF104647; Hydra Bcl-2-like 3: EU035765; Hydra Bcl-2-like
4: EU035764; Hydra Bcl-2-like 5: EU035763; Hydra Bcl-2-like 6:
EU035762; Hydra Bcl-2-like 7: EU035761
275
analysing sequences from the sea anemone Aiptasia [51].
The incomplete conservation of bcl-2 sequences in Hydra
and sea anemone made it questionable whether these cni-
darian Bcl-2 proteins were involved in programmed cell
death. However, our recent experiments have now shown
that six Hydra Bcl-2-like proteins inhibited apoptosis when
expressed in mammalian cells. One Bcl-2-like protein
mildly induced apoptosis. Two more sequences are very
similar to mammalian Bak-proteins (they were termed
HyBak-like 1 and 2) and strongly induced apoptosis in
human cells. We also showed that most of the Hydra Bcl-2
proteins are localised to mitochondria and that this locali-
sation is mediated via a C-terminal hydrophobic domain
[57]. Two, however, were localised to the ER. We there-
fore suggest that Hydra Bcl-2 proteins are involved in
apoptosis regulation and that apoptotic pathways involve
both mitochondria and the ER [53].
In mammals, a large family of BH3-only proteins is
involved in apoptosis induction. These include Bad,
PUMA, NOXA and many more [49]. Most of these pro-
teins are not related in primary sequence except for the
presence of the BH3 domain. BH3-only proteins of this
group have not been found in Drosophila. However, the
BH3-only protein Egl-1 in C. elegans is upregulated in all
somatic cells that are destined to die [58]. In addition, it is
upregulated after genotoxic insult in the C. elegans germ
line as a response to transcriptional activation by the
C. elegans p53 homolog [59]. In pre-bilaterian animals no
information about BH3-only proteins is currently available.
Our searches of the Hydra genome have now revealed the
presence of several genes encoding putative BH3-only
proteins. Investigations are underway to show where these
BH3-only proteins are expressed in Hydra and whether
they participate in apoptosis regulation. These BH3-only
proteins may hold an important key to understanding how
apoptosis is induced in Hydra.
Hydra insulin signalling in apoptosis regulation
Results discussed at the beginning of this review suggested
that apoptosis is used to ensure cellular homeostasis in
Hydra, especially under conditions of varying nutrition.
Sensing of metabolic activity and processing of this
information in order to adjust growth rate is achieved by
insulin signalling in many organisms. In mammals insulin
regulates glucose uptake, glycogen production and fat
storage by acting as an endocrine hormone. In inverte-
brates, insulin signalling also regulates growth [60]. The
picture here is very complex because Drososphila and
Caenorhabditis have a large number of insulin-like pep-
tides. C. elegans responds to food limitation by dauer
formation and this is regulated via insulin pathways
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276
Hydra proinsulin 1 rescues cells from apoptosis
70
HyFoxO + HyIns1 HyFoxO + RFP
60
50
% apoptotic cells
40
30
20
10
0 References
Fig. 5 HydraInsulin 1 (HyIns1) rescues cells expressing HyFOXO-
GFP from apoptosis. The HyFOXO-GFP sequence was cloned into
the vector HoTG. The HyProinsulin 1 sequence was cloned into the
HoTG vector after removal of the GFP encoding sequence; this leads
to expression of Hyproinsulin1 without the GFP-tag; its expression in
hydra cells was confirmed with an anti-proinsulin1 antibody.
Plasmids were transfected into hydra cells with the particle gun.
GFP expressing cells were scored as normal or apoptotic based on
nuclear morphology. As a control HyFOXO-GFP was expressed
together with RFP. The plasmid containing the HyProinsulin1 cDNA
(Accession number: GU219979) was a kind gift from Rob Steele
(Irvine)
including PI(3)kinase, PKB and the transcription factor
FoxO, which translocates to the nucleus in the absence of
insulin signals and induces gene expression that leads to
dauer formation [61]. Drosophila growth is also dependent
on food availability and is also regulated by insulin sig-
nalling through the insulin receptor, IRS, PKB and FoxO
[62]. Prerequisites for insulin signalling are also known for
Hydra. An insulin receptor homolog was cloned [63] and
three insulin genes have now been identified (Steele, per-
sonal communication). It was shown that protein kinase B
in Hydra has all the characterisitics to be regulated by
PI(3)kinase signalling [64] and that the PI(3) kinase
inhibitor wortmannin induces massive apoptosis in Hydra
[65]. When a Hydra FoxO-GFP fusion protein is expressed
ectopically in Hydra epithelial cells, it is partially trans-
located into the nucleus due to the GFP-tag [57, 66]. As a
consequence apoptosis is induced. Interestingly, when we
co-expressed the Hydra proinsulin-1 gene in such cells,
apoptosis was inhibited (Fig. 5). These preliminary results
constitute the first direct evidence that insulin signalling in
Hydra is involved in apoptosis regulation.
Perspectives
Based on the elaborate genetic machinery available for
apoptosis in Hydra and our initial experimental data on
123
Apoptosis (2010) 15:269–278
apoptosis in Hydra it appears that the network for the regu-
lation of apoptosis in pre-bilaterian animals was very com-
plex. Essentially all pathways that we know to be involved in
apoptosis regulation in mammals had already evolved in the
common ancestor of bilaterians and cnidarians (Table 1).
Hence, the simple cnidarian Hydra is emerging as a useful
model organism to study new aspects of apoptosis as a reg-
ulator of cellular homeostasis. The completion of the Hydra
genome sequencing in addition to new techniques to create
transgenic Hydra provide a substantial experimental plat-
form which will stimulate research in this area [67].
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