Molecular Cell Biology, Darmstadt University of Technology, Schnittspahnstraße 10, 64287 Darmstadt, Germany
* Authors for correspondence (e-mails: technau{at}bio.tu-darmstadt.de and holstein{at}bio.tu-darmstadt.de)
Accepted 2 October 2003
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SUMMARY |
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Key words: Hydra, Nematocyte, Neural development, Neuronal differentiation, Zic, odd-paired, achaete-scute, Nowa
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Introduction |
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Zic genes form a small family of Zn-finger transcription factors closely
related to the Gli-family (Aruga et al.,
1996a). Both are members of the superfamily of Krüppel-like
Zn-finger genes, which all have a conserved H/C link in common
(Schuh et al., 1986
). So far,
five Zic genes have been isolated from mouse, and six Zic genes have been
isolated from the frog, of which three, zic1, zic-r1 and
opl, are allelic to each other
(Aruga et al., 1994
;
Aruga et al., 1996a
;
Aruga et al., 1996b
;
Nakata et al., 2000
). The
sequences of these paralogs are highly similar to each other, indicating that
they arose by recent duplication events. In vertebrates, all paralogs have
overlapping expression patterns and appear to act cooperatively in neural and
neural crest induction (Aruga et al.,
1996a
; Nakata et al.,
1997
; Nakata et al.,
1998
; Nakata et al.,
2000
; Brewster et al.,
1998
; Nagai et al.,
1997
; Salero et al.,
2001
). Zic genes are expressed in the dorsal neuroectoderm, as
well as in the dorsal somitic mesoderm
(Aruga et al., 1996a
;
Grinblat and Sive, 2001
;
Nakata et al., 1998
;
Rohr et al., 1999
). Analysis
of Bmp and chordin mutants in zebrafish showed that the dorsal
restriction of zic2 expression requires Bmp signalling and the Bmp
antagonist Chordin, suggesting that Bmp2/4 inhibits Zic expression ventrally
(reviewed by Mayor and Aybar,
2001
; Rohr et al.,
1999
). By contrast, Sonic hedgehog, suppresses Zic expression in
the ventral neural tube and adaxial mesoderm
(Rohr et al., 1999
). Although
Zic genes have a conserved role in neurogenesis in all vertebrates,
Zic-related genes in lower chordates, the ascidians, appear to have additional
functions in muscle development, and are required for the formation of the
notochord and anterior mesenchyme (Nishida
and Sawada, 2001
; Satou et
al., 2002
; Imai et al.,
2002
; Wada and Saiga,
2002
). Strikingly, a role in neural development for the
Drosophila homolog odd-paired (opa) has not been reported.
Instead, opa acts as a pair-rule gene in epidermal segmentation
(Benedyk et al., 1994
), and
also has a role in the formation of midgut constrictions mediated by the
visceral mesoderm (Cimbora and Sakonju,
1995
). Hence, it is not clear whether the proneural function of
Zic in vertebrates represents the ancestral state of this gene or whether it
has been newly recruited to this function.
The only way to distinguish between the two possibilities is to analyze an
outgroup, i.e. a more basal group. The first nervous system in animal
evolution developed in the simple metazoan Cnidaria. However, it is unclear at
present whether the nervous systems in Cnidaria and Bilateria evolved
independently or whether they are homologous types of tissue. In most
cnidarian species like Hydra, the cnidarian nerve net is diffuse, but
with higher densities at particular positions. However, no brain-like
structures have been reported in Cnidaria. Interestingly, besides nerve cells,
there is another neuronal cell type in Cnidaria, the nematocytes or stinging
cells, a cell type exclusively found in Cnidaria. Nematocytes contain a large
single vesicle with a large capsule, the nematocyst. Nematocysts are extrusive
organelles, i.e. they can discharge and evaginate a tube or filament, often
attached with poisonous proteins. Most nematocytes are located in the
tentacles, where they are used by the animals to catch their prey. Nematocytes
are in contact with the environment via a cnidocil apparatus, a chemosensory
structure resembling the hair sensillum of the inner ear, and
electrophysiological studies have demonstrated that they have all the
properties of sensory cells (Hausmann and
Holstein, 1985; Brinkmann et
al., 1996
). Furthermore, in the freshwater polyp Hydra,
nematocytes and nerve cells both derive from the same pool of pluripotent stem
cells (David and Gierer, 1974
;
David and Murphy, 1977
).
Hence, nematocytes are regarded as a second neuronal cell type in
Cnidaria.
The molecular regulation of neuronal differentiation in Hydra is
largely unknown. So far, only one of the genes involved in bilaterian
neurogenesis has been isolated from Hydra, the bHLH transcription
factor gene achaete-scute homolog Cnash
(Grens et al., 1995).
Interestingly, Cnash is not expressed in nerve cells or nerve cell
precursors, but in cell clusters that give rise to nematocytes, consistent
with the idea that nematocytes are a neuronal cell type.
We now report the isolation and characterization of a homolog of the
zic/opa gene in Hydra. We show, that it is also expressed in
the nematocyte lineage, and that it acts upstream of Cnash and
Nowa, a differentiation gene of all nematocytes
(Engel et al., 2002). First
expression can be detected at the level of stem cells and is restricted to the
proliferative stages of nematocyte differentiation. It is therefore the
earliest marker gene for the differentiation of a neuronal cell type in
cnidarians. The evolutionary implications are discussed.
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Materials and methods |
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Elimination of interstitial cells
To eliminate interstitial cells, the temperature-sensitive mutant strain
sf-1 was cultured at 26°C for up to three days
(Sugiyama and Fujisawa, 1978;
Terada et al., 1988
).
Alternatively, the wild-type strain Basel of H. vulgaris was treated
for up to three days with 10 mM hydroxyurea (HU). In both cases animals were
fed daily during the treatment. Maceration analysis
(David, 1973
) confirmed that
95% of all interstitial cells had disappeared after one day (data not
shown).
Cloning of Hyzic
A full-length clone of Hyzic (GenBank Accession Number AY436645)
was isolated by screening a Hydra cDNA library with the degenerate
oligonucleotide ACNCAYACWGGWGARAARCCWTT, directed against the conserved H/C
link (THTGVKPF) of Krüppel-like Zn-finger proteins
(Schuh et al., 1986).
Hybridization was carried out following standard protocols
(Sambrook et al., 1989
).
Briefly, hybridization was carried out overnight at 42°C. Filters were
then washed twice at room temperature in 6xSSC/0.05% Na-Pyrophosphate
(Na-PPi) for 15 minutes, once in 3 M TMAC at room temperature, twice in 3 M
TMAC at 42°C, twice in 3 M TMAC at 47°C, and once in 6xSSC/0.05%
Na-PPi for 15 minutes at room temperature, before being developed by
autoradiography. Double-positive clones were isolated by the Lambda Zap II in
vivo excision system (Stratagene) and sequenced. As the isolated clone did not
contain stop codons in all three reading frames at the 5' end, we
performed 5'RACE using the Gene Racer kit (Invitrogen). We isolated a
short RACE clone encoding an additional 119 bp with stop codons in all the
reading frames. Accordingly, the first AUG in the library clone putatively
encodes for the translation start site.
Phylogenetic analysis
The Maximum Likelihood program TREE-PUZZLE
(Schmidt et al., 2002) was
used to perform a phylogenetic analysis. JTT was used as substitution model,
the rate heterogeneity parameter alpha of the gamma distribution was
calculated from the data set. 10,000 replica were calculated. Similar tree
topologies were obtained by using full-length protein sequences or the
Zn-finger domain alone.
Single and double whole-mount in situ hybridization
Whole-mount in situ hybridization using single, FITC- or DIG-labeled RNA
probes was carried out as described previously by Grens et al.
(Grens et al., 1995), except
that NBT/BCIP (Roche) was used as a substrate for staining. The
double-labeling in situ hybridization procedure was carried out according to
the protocol described by Hansen et al.
(Hansen et al., 2000
). The
Hyzic probe was labeled with FITC, the Nowa probe was
labeled either with DIG or FITC, and the Cnash probe was labeled with
DIG. The first round of staining was always carried out with Fast Red
substrate, irrespective of whether the probe was FITC or DIG labeled.
Accordingly, the detection of Cnash was always carried out with
NBT/BCIP as substrate, as Cnash expression is weaker than that of
Hyzic or Nowa. All photographs were taken using either a
digital Spot Camera (Diagnostic Instruments) or a Hamamatsu Digital Camera
(Hamamatsu Photonics), controlled by the MetaMorph and Hipic Software,
respectively. Some pictures were false-colored in Photoshop.
BrdU/in situ hybridization double staining on whole-mounts
Prior to in situ hybridization, polyps were treated with BrdU (5 mM in
Hydra medium, HM) for 60 minutes, followed by two washing steps in HM
and fixation with 4% paraformaldehyde (PFA) overnight. After staining with
NBT/BCIP for the labelled RNA probe and destaining for 30 minutes in 100%
ethanol, animals were washed in PBS four times for 10 minutes. Then, animals
were incubated for 30 minutes in 2 N HCl and washed 4 times in PBS. An
anti-BrdU monoclonal antibody (Roche) was added in a dilution of 1:15 in
PBS/1% BSA/0.1% Tween 20, overnight at 4°C. Excess antibody was removed by
four 15-minute washes in PBS before incubation for 3 hours at room temperature
with anti-mouse antibody coupled to ALEXA-488 fluorochrome (Molecular Probes),
used in a dilution of 1:400 in PBS/1% BSA. Finally, animals were washed three
times in PBS for 10 minutes, mounted in PBS/glycerol (1:9) and examined under
the Zeiss Axioscope 2 plus.
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Results |
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Hyzic is expressed in the nematocyte differentiation pathway
In Hydra, the pluripotent interstitial cell lineage can give rise
to nerve cells, nematocytes, gland cells and gametes (reviewed by
Bode and David, 1978).
Quantitatively, the two major derivatives are the two neuronal cell types,
nerve cells and nematocytes. Nerve cell precursor cells are committed
stochastically in the body column and migrate to the site of their final
differentiation, predominantly in the head and the foot
(Heimfeld and Bode, 1984a
;
Heimfeld and Bode, 1984b
;
Technau and Holstein, 1996
;
Hager and David, 1997
). By
comparison, nematocyte differentiation also begins stochastically in the body
column, but involves a varying number of synchronous cell divisions, leading
to nests of 4, 8, 16 and 32 nematoblasts. At any of these stages, nematoblast
nests can undergo a final mitosis and start differentiating the nematocyte
capsule (David and Challoner,
1974
; David and Gierer,
1974
). Hence, nematocytes differentiate as nests of 8, 16, 32 or
64 cells. When the formation of the capsule is completed, the nests separate
into single nematocytes. Most of them subsequently migrate into the tentacles
and are incorporated in the battery cells.
From whole-mount in situ hybridization (WISH) analysis it was determined
that Hyzic is expressed in the body column in cells of the ectodermal
layer. No Hyzic cells are found in the hypostome, the tentacles or
the lower peduncle and foot region (Fig.
2A). This expression pattern was reminiscent of that of the
achaete-scute homolog Cnash
(Fig. 2B)
(Grens et al., 1995), and of
Nowa, which encodes for a structural protein of the outer capsule
wall (Fig. 2C) (Engel et al., 2002
). This
indicated that Hyzic is also expressed in the nematocyte pathway, or
in another pathway of the interstitial cell lineage.
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Discussion |
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Hyzic is involved in nematocyte differentiation in Hydra
As nematocytes are sensory cells, they are regarded as a specialized
neuronal cell type that derives from the same pluripotent stem cell population
as nerve cells (David and Gierer,
1974; Bode, 1988
).
Cell cloning and labeling experiments have established that cell clusters of 4
cells or larger usually differentiate into nematocytes
(David and Gierer, 1974
;
David and Challoner, 1974
;
David and Murphy, 1977
;
Hager and David, 1997
). Our
analysis shows that Hyzic is, to date, the earliest marker of
nematocyte differentiation, starting at the level of the interstitial stem
cells. Hyzic expression is detected in the proliferative stages, and
includes single and pairs of interstitial cells (1s+2s), which are part of the
interstitial stem cell pool. Interestingly, the S-phase labeling index (40%)
is significantly lower than would be expected from previous measurements of
the cell cycle parameters of nematoblasts
(Campbell and David, 1974
).
This could indicate either an underestimation of the cell cycle length of
nematoblasts, or that Hyzic not only marks this population but also
includes other more slowly cycling cells or cells that have exited the cell
cycle. Consistent with the early expression, we find no overlapping expression
with the differentiation marker Nowa
(Engel et al., 2002
), which
codes for a major wall protein of the nematocyte capsule, H22, a protein that
is recognized by the monoclonal antibody H22
(Kurz et al., 1991
).
Nowa and H22 expression can be strongly detected after terminal
mitosis (Engel et al., 2002
).
Nowa mRNA is downregulated in mature nematocytes, but the protein can
still be detected.
Surprisingly, we also do not find an overlap of expression of
Hyzic with the achaete-scute homolog Cnash
(Grens et al., 1995), another
gene involved in nematocyte differentiation. This gene is expressed in nests
of differentiating nematocytes, but was also found to be expressed in
interstitial cells, as shown by macerate in situ hybridization
(Grens et al., 1995
). Hence,
Cnash expression is expected to span the time window from
proliferating interstitial cells resembling stem cells until differentiating
nematocytes with developing capsules
(Grens et al., 1995
). Although
we can not completely rule out the possibility that the difference between the
findings in this paper and those of Grens et al.
(Grens et al., 1995
) is due to
different sensitivities in macerate and whole-mount in situ hybridization, our
results show, that Cnash expression is restricted to
non-proliferating stages of the nematocyte differentiation pathway. Several
lines of evidence support this. (1) Following pulse labeling with BrdU, no
labeled Cnash cells could be detected, and the first BrdU-positive
Cnash cells appeared between 24 and 48 hours of continuous labeling
(Fig. 6E). (2) Elimination of
the proliferating interstitial cell population in the mutant strain
sf-1, or in HU-treated wild-type animals, did not affect the number
of Cnash expressing cells during the first 48 hours, whereas it
strongly affected the number of proliferating Hyzic cells
(Fig. 7G). (3) Cnash
shows substantial overlapping expression with Nowa
(Fig. 5). (4) The average
cluster size of Hyzic expressing cells is smaller by one cell
division compared with Cnash and the non-proliferating Nowa
cells, suggesting that Hyzic cells have gone through a final mitosis
and turned on Cnash expression
(Fig. 3F). (5) We recently
isolated a JNK gene that is also expressed in the nematocyte differentiation
pathway. Expression of this JNK gene overlaps both with Hyzic and
with Nowa, thereby bridging Hyzic expression and
Cnash/Nowa expression (I. Philipp, B. Hobmayer and T.W.H.,
unpublished). Hence, we conclude that Hyzic is expressed early,
primarily during the proliferative stages, while Cnash is expressed
later, after the final mitosis.
Does Hyzic mark an independent sublineage?
Because Hyzic and Cnash expression do not overlap, there
is still the possibility that the two genes mark two separate sublineages. In
fact, Hydra have four different types of nematocytes: stenoteles,
holotrichous isorhizas, atrichous isorhizas and desmonemes
(Holstein et al., 1990), and
one could imagine, that Hyzic is expressed only in one or few of
these nematocyte subtypes. At present, conflicting reports exist about whether
commitment to the different subtypes occurs early or late during nematocyte
differentiation (Fujisawa and David,
1981
; Fujisawa and David,
1982
; Shimizu and Bode,
1995
). Most Hyzic-expressing cell clusters consist of 4
or 8 cells, and we also detected few single interstitial cells expressing
Hyzic. Interestingly, in BrdU pulse-labeling experiments, we also
found nests of 4 and 8 cells that did not express Hyzic
(Fig. 6D). This could mean
either that Hyzic marks only one particular subpopulation of
nematoblasts, or that the onset of Hyzic expression is not sharply
defined to a particular stage. We favor the second interpretation for the
following reasons. (1) Pulse-labeling experiments reveal that up to 30% of all
BrdU-positive cell clusters (4 or 8 cell clusters) do not express
Hyzic. Thus, if the Hyzic-negative cells represented a
second independent lineage, we would expect that it makes up one third of all
developing nematocytes. This is highly unlikely. All nematocyte precursors
express Nowa (Engel et al.,
2002
), which encodes H22, a protein of the outer nematocyst wall.
Nowa expression temporally overlaps with that of Cnash,
which is consistent with the finding that Cnash is expressed in the
great majority of all differentiating nematocytes
(Grens et al., 1995
).
Furthermore, Cnash/Nowa expression and Hyzic expression are
bridged by the expression of the JNK gene (see above). Hence there is no
evidence for a second major subpopulation besides the Hyzic cells.
(2) At any cluster size, only a certain fraction keeps proliferating, while
the other fraction undergoes a final mitosis and starts differentiating into
nematocytes (see Fig. 8). Thus,
if Hyzic is turned on sharply at the transition between the 2-cell
and the 4-cell stage, we would expect less Hyzic-expressing 8-cell
clusters, and even less 16-cell clusters, and so on. However, we found only a
few 1s+2s, many 4-cell clusters, and almost as many 8-cell clusters,
expressing Hyzic (Fig.
3F); at the 16-cell cluster size the number drastically decreases.
It is an obvious possibility that Hyzic expression remains after the
division into the 8-cell cluster, irrespective of whether this cluster remains
in the cell cycle or whether it enters the differentiation pathway. This would
be consistent with the comparatively low BrdU-labeling index. However, this
should lead to an overlapping expression with Nowa, which is turned
on in all differentiating nematocytes immediately after the final mitosis
(Engel et al., 2002
). However,
we never observed such an overlap. This can be best explained by a continuous
supply of proliferating 4- and 8-cell clusters from the pool of
Hyzic-negative cell clusters, compensating for the loss of
Hyzic expressing clusters by differentiation. We therefore propose
that the onset of Hyzic expression is not sharply defined and can
occur between the 2-cell and the 8-cell stage.
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A possible role for Hyzic in nematocyte differentiation
Although all attempts to knockdown the Hyzic transcript by RNAi
failed (data not shown), our data are consistent with the view that
Hyzic marks the commitment to the nematocyte differentiation pathway.
The variable onset of Hyzic expression suggests that the commitment
can occur in a prolonged phase during proliferation of the interstitial cells.
Whether the Hyzic-negative cell clusters still have stem-cell like
properties, and whether they can still differentiate into neurons, is unclear.
It is well established that most neurons differentiate in pairs after a final
mitosis from individual interstitial cells
(Hager and David, 1997;
Heimfeld and Bode, 1984b
;
Holstein and David, 1986
).
However, some differentiation of 4 and 8 neurons from single migrating
interstitial cells has occasionally also been observed
(Heimfeld and Bode, 1986
;
Hager and David, 1997
),
indicating that they underwent one or two additional rounds of mitosis. Yet, a
neuronal differentiation from the cycling Hyzic-negative cell
clusters would require a breakdown into single cells, because 4- and 8-cell
clusters were never observed as intermediate stages in nerve cell
differentiation. Furthermore, committed precursor cells of the interstitial
cell lineage might exist that have differing capacities for proliferation,
allowing for the regulation of amplification rounds
(Heimfeld and Bode, 1986
).
Thus, the likelihood to differentiate into neurons decreases with the cluster
size, whereas the probability to differentiate into nematocytes increases with
the cluster size (see Fig. 8A).
If this was the case, we would expect to find genes that are expressed in the
non-committed interstitial cells and in few nests of 4 cells and 8 cells (see
above).
What could be the role of Hyzic in nematocyte differentiation?
Because it starts being expressed in interstitial cells, it might commit stem
cells to the nematocyte pathway. Interestingly, in mice, Zic1 is
expressed in proliferating neuronal precursors, but inhibits terminal neuronal
differentiation (Aruga et al.,
2002a). Consistently, Zic1 positively regulates levels of
Cyclin D, and inhibits the mitosis inhibitors p27 and p16
(Aruga et al., 2002b
). Hence,
overexpression or ectopic expression of Zic1 leads to an expansion of
dorsal neurons due to extended rounds of proliferation (reviewed by
Bally-Cuif and Hammerschmidt,
2003
). This is very similar to the situation in Hydra,
where Hyzic might keep cells in the cell cycle that are committed to
the nematocyte pathway, thereby ensuring that large numbers of nematocytes can
differentiate. Hydra has a turnover of several thousand nematocytes
per day; hence, proliferation of nematocyte precursors is primordial in order
to amplify the number of nematocytes.
Evolutionary considerations
Neural development is largely conserved from insects to vertebrates on the
molecular level (reviewed by Sasai,
2001). The Bmp antagonist Sog/Chordin defines the neurogenic
region high up in the molecular hierarchy. As a result, proneural genes of the
bHLH family (achaete-scute, atonal/neurogenin) are activated, which
further activate the neurogenic genes Notch and Delta. In
vertebrates, the zinc finger gene Zic, is activated downstream of
Chordin and upstream of neurogenin
(Mizuseki et al., 1998
;
Kuo et al., 1998
); hence, it
acts very early in the hierarchy of neural differentiation, in particular in
the differentiation of the neural crest. Yet, the Drosophila homolog,
odd-paired (opa) is not involved in neural differentiation at all. It
rather has a pair-rule function in segmentation and in midgut constriction
(Ingham et al., 1988
;
Benedyk et al., 1994
;
Cimbora and Sakonju, 1995
).
Thus, it is unclear, whether the function of vertebrate Zic genes in neural
differentiation is ancestral or newly adopted. Interestingly, we also recently
identified a Hydra Sog/Chordin ortholog (F. Rentzsch and T.W.H.,
unpublished), and the situation in a member of an outgroup to the Bilateria,
such as the cnidarian Hydra, is therefore of great significance for
this question.
In vertebrates, Zic is downstream of Chordin, and epistatic to the
proneural bHLH genes achaete-scute and neurogenin
(Mizuseki et al., 1998). In
zebrafish, as well as in the lower chordates Amphioxus and Ciona,
homologs of the Zic family are also expressed in specific domains of the
dorsal mesoderm, in particular in parts of the somites
(Gostling and Shimeld, 2003
).
On the basis of these results, the mesodermal expression has been proposed to
reflect the ancestral situation for all chordates
(Gostling and Shimeld, 2003
).
Although the role of Zic in this tissue is not understood, it could reflect
the influence of Chordin in dorsal compartments of the embryo. In
Hydra, because Hyzic is expressed in the proliferating
stages of a neuronal cell type, the nematocytes, we propose that the ancestral
function of Zic was in neuronal differentiation. Furthermore, like in
vertebrates, Hyzic acts upstream of the proneural bHLH gene
Cnash, an achaete-scute homolog
(Fig. 8B,C)
(Grens et al., 1995
). This
suggests that components of the gene cascades regulating the differentiation
of neuronal cell types are conserved from Hydra to vertebrates.
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ACKNOWLEDGMENTS |
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