VIENNA

The role of Wnt signaling in axis development and stem cell

regulation in Cnidarians
Elias Celso Theodor DE CILLIA1
1 University of Vienna Masterstudent Genetik und Entwicklungsbiologie

∗ Correspondance: a12114535@unet.univie.ac.at
This work was created as part of the practical course 300302 UE Übung in Entwicklungsbiologie (2022W).

Abstract
Cnidarians, the sister group of bilaterians, have been a useful model organism in understanding the evolutionary origins of bilaterians, and for
gaining insights into the diversity of animal body plans and developmental processes. Here the two cnidarians Nematostella vectensis and
Hydra vulgaris are used in a series of experiments centered around the Wnt signaling pathway and its role in axis development as well as stem
cell regulation. Here we show that the ectopic activation of Wnt signaling in Nematostella leads to the expansion of the expression pattern of
the gland marker pry, and its disappearance in the case of the fibroblast-growth-factor fgfa1. Furthermore, cell proliferation is shown to not be
significantly altered when ectopic Wnt signaling is activated. Additionally, the role of axial patterning was further investigated in the context of
single-cell suspension aggregates, although results showed to be inconclusive. Moreover, we show that the migration of stem cell derivatives
towards the foot of Hydra vulgaris is significantly increased after stem-cell depleting hydroxyurea treatment is administered, with no changes
observed for the cell migration towards the head.

Keywords: Wnt; Cnidarian; Axis deveolpment.

Introduction cell surface receptors and initiate a cascade of intracellular

Cnidarians are a diverse group of animals that include jellyfish,
coral, and sea anemones. They are some of the most primitive
animals with a nervous system and exhibit radial symmetry.
Recent research in molecular biology and phylogenetics has
revealed that cnidarians are the sister clade of Bilateria, a
group of animals that includes all the familiar animals with
bilateral symmetry such as insects, reptiles, and mammals
(Genikhovich and Technau 2017)(Whelan et al. 2015). This
means that cnidarians and bilaterians share a common ancestor
that diverged over 600 million years ago (Hejnol et al. 2009).
The recognition of cnidarians as the sister clade of Bilateria
has important implications for understanding the evolution
of animal diversity. It suggests that bilateral symmetry and
the associated body plan innovations seen in bilaterians, such
as the development of organs and complex nervous systems,
evolved independently of the ancestral radial symmetry seen in
cnidarians. Furthermore, the study of cnidarians can provide
insights into the early evolution of animal body plans and
the genetic and developmental mechanisms underlying the
transition from radial to bilateral symmetry. Despite their simple
body plans, cnidarians have a complex network of signaling
pathways that control various developmental processes. One
such pathway is the Wnt signaling pathway. It is a highly
conserved signaling pathway that plays a crucial role in
embryonic development, axial patterning (Onai 2018) stem cell
maintenance (Khalturin et al. 2007), and tissue regeneration
(Clevers et al. 2014) in many animals. The pathway is named
after the Wnt family of secreted glycoproteins that bind to
signaling events. It is initiated by the binding of Wnt ligands
to Frizzled (Fzd) receptors and co-receptors, such as LRP5/6,
on the cell surface. This binding leads to the activation of
Disheveled (Dvl) protein, which inhibits the activity of the
β-catenin destruction complex, consisting of Axin, APC, and
GSK3β. As a result, β-catenin accumulates in the cytoplasm and
translocates to the nucleus, where it interacts with members
of the TCF/LEF transcription factor family to activate the
transcription of target genes involved in cell proliferation,
survival, and differentiation. A well-established way of ectopic
activation is the usage of GSK3β inhibitors such as alsterpaullone
or azakenpaullone. Additionally, knockouts of the destruction
complexes components also lead to an accumulation of β-
catenin, therefore presenting a viable alternative for ectopic wnt
signaling activation. In this work, both the azakenpaullone
treatment and APC-mutant will be used. In cnidarians, the
Wnt pathway has been shown to play a role in axial patterning
and differentiation of cells during embryonic development. The
study of Wnt signaling in cnidarians is important because it
provides insights into the evolution of this pathway and its role
in animal development. Additionally, cnidarians are valuable
models for studying the function of Wnt signaling due to their
simple body plans and relatively easy manipulability. In this
study, the anthozoan Nematostella vectensis and the hydrozoan
Hydra vulgaris were chosen as model organisms. As axial
patterning was of special interest for this work, a variety of
transcription factors and signaling pathway components that are
expressed in early embryonic development were studied. They
2 300302 UE Übung in Entwicklungsbiologie (2022W)
can be classified as oral, aboral, midbody and gland markers.
The forkehead-box transcription factor foxa1, as well as the T-
box transcription factor brachyury, are both oral markers, first
expressed during late blastula stage as a ring of cells marking the
blastopore. Expression stays around the blastopore during early
gastrulation and remains expressed in the pharynx in primary
polyps (Fritzenwanker et al. 2004) (Scholz and Technau 2003). As
part of the WNT gene family, wnt2 is found to be expressed as a
medial band during early embryonic development (Marlow
et al. 2013), thus, classifying it as a mid-body marker. The
fibroblast-growth factor fgfa1 is expressed in the apical organ of
Nematostella vectensis, it´s expression starts at the early planula
at the aboral pole and remains there throughout early embryonic
development up until the primary polyp stage (Rentzsch et al.
2008). Since WNT signaling is not also important for axial
patterning during early development but also for stem cell
proliferation and differentiation transplantation experiments
using Hydra were performed to further investigate stem cell
migrational behavior. As Neamtostella vectensis is known to
secrete a mucus-like material from the wound (DuBuc et al.
2014) the transplantation experiments would not be possible,
as mucus would keep the graft and host from being in close
contact, which is necessary for the transplantation to heal. Thus
the switch to another cnidarian, namely the hydrozoan Hydra
vulgaris was made in which the transplantation works well, due
to no mucus being produced upon dissection In Hydra vulgaris,
ectopic Wnt activation has been shown to directly inhibit the
differentiation of interstitial stem cells into derivatives, i.e.
nematoblasts (Khalturin et al. 2007). Here we used hydroxyurea
(HU) treatment to deplete the stem cell population of Hydra
vulgaris, to study the mechanism of regeneration in Hydra. To
visualize stem cells as well as their derivatives, a transgenic line
was used, in which the stem cell-specific promotor cnnos1 drives
the expression of a green fluorescent protein and the action
promoter drives the expression of a red fluorescent protein.
Transplanting the transgenic grafts onto HU-treated hosts allows
monitoring of the behavior of stem cell niches during increased
demand for cell regeneration.
Materials and methods
Nematostella culture
Animals were maintained in 0.3× filtered seawater (NM). Eggs
were fertilized by transferring them into sperm containing NM,
then left undisturbed for 30 to 60 min. Jelly packages were
removed by incubation for 25 min in 3% cysteine/ NM. Embryos
were washed with fresh NM and then raised at 21°C.
Hydra culture
The animals were cultured in “Hydra-medium,” which was
adjusted to pH 7.8 and daily fed with Artemia brine shrimps.
All animals used in the experiments were starved for 24 hr prior
to the experiment.
RNA probe synthesis for foxA1, brachyury, wnt2, fgfa1,
pry isolated from Nematostella
NA extraction was performed using embryos of different stages
following standard protocol, bulk RNA was transcribed into
cDNA (with final concentrations of random hexamers 2,5µM;
RNA 5 µg total amount; dNTP mix 500 µM; Dithiothreitol 5 µM;
RNAse OUT 2 U/µl; Protoscript II 10 U/µl). foxA1, brachyury,
wnt2, fgfa1 and pry gene sequences were amplified using gene-
specifies forward and reverse primer (with final concentrations
of fwd-primer and rev-primer 1 µM; ddNTPs 200 µM; Q5 buffer
1x; Q5 polymerase 0,04 U/µl). The PCR product was run on
an agarose gel to confirm the expected PCR fragment sizes.
After gel band purification using peqGold purification kit vector
ligation was performed (with final volumes of 2x Rapid Ligation
buffer 2,5 µl; pJET Vector 0,5µl; PCR product 1,5µl; T4 DNA
ligase 0,5µl).Transformation of competent E. coli bacteria was
performed followed by plating on ampicillin-agar plates and
incubating at 37°C overnight. Clones were picked and PCR
was performed to check the insert direction. Clones with anti-
sense insert were selected and transferred to 5ml ampicillin
containing Supermedium and left on a shaker at 37°C overnight.
Mini prep was performed using AnalytikJena miniprep kit.
PCR was performed to amplify the probe template (with final
concentrations of 10x Dreamtaq buffer 1x; dNTPs 150 µM;
primer pJET_fw_t7_outer 0,5 µM; primer pJET_Rv_Sp6 0,5 µM;
Tay Polymerase 0,02 U/µl). Megascript (Ambion) Dig-labeled
RNA synthesis was performed ( with final concentrations of
T7 or SP6 transcription buffer 1x; PCR maximum; DIG-UTP
labeling mix 1x; RNAse OUT 2 U/µl; T7 or SP6 enzyme mix
1,5x).
Inhibitor Treatment
The GSK3β inhibitor Azakenpaullone (AZK) was applied at
final concentrations of 2 µM in 0.1% DMSO. Control animals
were incubated at the same concentration of 0.1% DMSO only.
Solutions were applied for 24 hours and then exchanged for 0.5%
DMSO to improve in situ hybridization results.
Hydroxyurea Treatment
The i-cell depleting reagent hydroxyurea (HU) was applied at
final concentrations of 5mM for 3 days and then left to recover
for 5 days and 10mM for 3 days and 1-day recovery. Animals
were left to recover in fresh Hydra medium.
Generation of single-cell suspensions
Gastrulae at 24h postfertilization were collected in 0.5 mL
Eppendorf tubes which had been pre-coated with 0.1% PBT
in order to avoid sticking cells to the tube. Double the volume of
filtered Calcium Magnesium free seawater at 14 ‰ was added
before vigorously pipetting with cut tips for 2-4 min. 1ml of
NM was added and cells were spun for 4 mins at 400 crf. After
resuspension in PBS cells were spun a 2nd time for 4min at
400crf. After resuspension in 110 µl PBs 10 µl of live/dead stain
was added and concentration and viability were counted. Cell
concentrations were adjusted to obtain cell aggregates of 10K,
50K and 100K cells and pipetted into 96-well plates which were
spun down at 250g for 5 minutes to form aggregates. Aggregates
were then incubated at 21°C and cleaned with fresh NM after
24h post aggregation.
Cell proliferation assays
Embryos were incubated with 30 µM EdU/DMSO in NM for
30 min at 21°C and then fixed immediately with 4% PFA for
1h. EdU incorporation was visualized by adding a fluorophore
to the Edu through a “click” chemistry reaction, then analyzed
under a fluorescent microscope.
In Situ Hybridization and Immunostaining
Hier muss ich noch etwas über ISH schreiben
 De Cillia et al. 3

Hydra transplantations
Large bud-less transgenic animals were selected and dissected
in the middle of the body then pushed, wound side first, onto a
piece of fishing line. A small incision of the basal disc is needed
to create an opening for the fishing line. Nontransgenic animals
were then cut accordingly and pushed onto the same fishing line
to form a new animal. Two pieces of parafilm pushed on the
fishing line from both sides keeps the two wounds in contact.
Animals were left to heal for 1-2h on fishing line, then removed
and kept in normal NM to recover from transplantation.

Results
Expression patterns in Nematostella
The examination of the expression pattern of 5 transcription
factors and signaling pathway components in Nematostella
vectensis visualized through in situ hybridization showed
4 spatially distinguishable expression zones. Foxa1 and
brachyury’s expression domains are virtually indistinguishable,
they both mark the future blastopore and mark the mesenteries
in older animals (Reference to forkhead paper) due to this they
can be used as markers for the oral pole of the embryos (Fig.
1A). Wnt2 showed to be expressed in a medial band between the
oral and aboral poles, most prominent in the 24h old embryo.
In the primary polyp stage, wnt2 expression was detected in
the mesenteries (Fig. 1B). The FGF ligand fgfa1 showed to be
Figure 1 Gene expression during Nematostella vectensis
expressed at the apical pole of the embryo, having a broader
development.
disc-like pattern in the early 24h stage which is reduced to a
single dot as the embryo moves through the developmental Spatial expression of both oral markers foxa1 and brachyury (A)
stages. In the primary polyp, no fgfa1 expression was detected Gene expression pattern for midbody marker wnt2 (B) and
(Fig. C). Thus, making fgfa1 an excellent candidate as an aboral aboral marker fgfa1 (C). Gene expression pattern of gland
pole marker. The expression pattern of pry starts as a spotted marker pry (D).
pattern equally distributed along the embryo in stage 24h, with
more pry-expressing cells accumulating at the aboral pole in
stages 48h to 72h (Fig. 1D).
The effects of ectopic activation of the canonical wnt signaling the DMSO control, the AZK-treated polyps (Fig. 3B-C) do not
pathway can only be observed in 2 of the 5 tested transcription show a distinguishable difference in the EdU signal brightness
factors. When looking at both the Azakenpaullone (AZK) and localization.
treated embryos and the APC knock-out mutants, no observable
differences in expression pattern were detected for foxa1, Single-cell suspension aggregates data showed to be
brachyury and fgfa1. In comparison to the DMSO control inconclusive
foxa1 and brachyury had the same oral and fgfa1 the same The in-situ hybridization stainings did not show a clear
aboral expression pattern (Fig. 2A). The transcription factor relationship between the number of oral-aboral axes formed
wnt2 showed no expression upon AZK treatment in the 72h old after aggregation and the initial size of the single-cell suspension
embryo, the medial band observed in the DMSO control was aggregates. Although the oral marker gene foxa1, known to
completely lost (Fig. 2B). Contrary to that, the pry expression stain the blastopore, did show multiple expression zones on the
pattern expanded into the whole embryo after ectopic Wnt aggregates (Fig. 4-10K,50K) as well as the aboral marker six3/6
activation through both the AZK treatment and APC knockout (Fig. 4-10K), overall results weren’t robust enough to quantify
(Fig.2C). the ration between initial size and formed axis.

Cell proliferation assay did not show increased cell
division in AZK-treated primary polyps
To further investigate the effects of ectopic activation of Wnt
signaling on cell proliferation, we incubated primary polyps
in presence of the thymidine analog EdU. Note that, no
quantification method was used to quantify the EdU signal
against the DAPI staining, visualizing the cell nucleus. Thus,
drawing any conclusion from the data of the cell proliferation
assay, represented in Figure 3, cannot be confidently made.
The EdU signal is most prominent at the oral pole in the area
around and under the pharynx, but also EdU-incorporated cells
scattered across the polyp were detected. In comparison with
Depletion of i-cells resulted in an increased migration
rate of derivatives in Hydra vulgaris
The transplantation of transgenic grafts onto HU treated host
showed, that the hydroxyurea-mediated depletion of interstitial
stem cells affected the migration of i-cell derivatives toward
the foot but not the head. Furthermore, HU treatment was
successful in increasing cell migration, as the transplantation
site became less clear and distinguishable after treatment (Fig. 5.
B,C) when compared to the sharp boundary between host and
graft tissue, observed in the control treatment (Fig. 5. A) The
HU treatment was able to increase the number of cells harboring
a fluorescent signal found in the host tissue. The border marking
4 300302 UE Übung in Entwicklungsbiologie (2022W)
Figure 2 Wnt overexpression in 72h old embryo through AZK
treatment and APC ko.
Unchanged gene expression pattern of foxa1, bra and fgfa1 after
ectopic Wnt signaling activation through Wnt agonist
Azakenpaullone treatment and APC knock-out mutant (A).
Expressing pattern of midbody marker wnt following ectopic
Wnt activation (B). Expression pattern expansion of the gland
marker pry following ectopic Wnt activation (C).
graft and host tissue was very clear in the control transplant,
with little to no fluorescent cells found in the non-transgenic
host tissue at the transplant site (Fig. 5A) and the foot (Fig. 6B).
However, the untreated host heads showed an increased number
of fluorescent cells compared to the foot (Fig. 6 A,B), showing
that even without stem cell depletion the migration direction
of stem cells derivates towards the head seems to be stronger
than towards the foot, which can be account for by considering
the primary mode of growth of Hydra. Upon HU treatment,
both concentrations were successful in increasing the number
of derivatives originating from the graft noticeably in the foot
(Fig. 6 D,F) but not in the head, as fluorescent cell numbers were
already high there in the control sample (Fig. 6 C,E).
Discussion
In this study, the spatial expression of five transcription factors
and signaling pathway components were examined. Ectopic
activation of Wnt signaling, through Azakenpaullone treatment
and APC knock-out, led to changes in expression patterns
in 2 out of 5 studied genes, namely fgfa1 and pry. In fgfa1,
ectopic activation was followed by the absence of an expression
pattern whereas the expression pattern of pry, expanded into
the whole embryo following AZK treatment or APC knockout.
Since this study was conducted as a part of a practical course
in developmental biology, some data has been lost, or not
Figure 3 Cell proliferation was not increased by ectopic wnt
signaling activation in Nematostella vectensis primary polyps.
EdU staining of 9-day-old Nematostella vectensis primary polyp
in lateral view after DMSO control treatment (A). Oral views of
EdU stained 9-day-old primary polyps after Azakenpaullone
incubation (B,C).
Figure 4 Oral and aboral pole markers in single cell
suspension aggregates.
In situ staining of oral marker foxa1 and aboral marker six3/6
for single cell suspension aggregates of differing initial sizes.
all conditions were imaged therefore leading to missing data.
This makes the interpretation of the results challenging, as the
absence of any effect of AZK and APC-mutants on the expression
pattern for foxa1, brachyury and wnt2 may have been caused
by a carryover from the untreated control into the treatment or
mutants, or incorrect naming of files during image acquisition.
The same comments can be made when examining single-cell
suspension aggregate data. As the single-cell suspension was
not flawless for all groups, and some cell clusters were still
present when aggregation took place, the resulting data may not
be significant. As the idea of the experiment was to remove any
cell organization by disrupting the cell-to-cell communication
through single-cell suspension, leftover cell clumps would have
kept their organization and therefore made the comparison
between initial aggregate sizes difficult.
Transplantation experiment of transgenic grafts onto i-cell
depleted hosts, mediated through hydroxyurea treatment,
showed to affect the migration of cells origenating from the
 De Cillia et al. 5

Figure 5 Differences in transplantation border after hydroxyurea treatment in Hydra vulgaris.

Transplantation of transgenic graft tissue onto non-treated control host showing sharp transplantation border (A). Increased cell
migration into host tissue after 10mM HU (B) and 5mM HU treatment (C).

graft towards the host. More precisely, the number of cells that of actin expression can start before the GFP is fully degraded,
migrated from the graft tissue into the host tissue was very low leading to a cell that harbors both fluorescent protein types.
in the foot of the host, but very high in the head, when no HU
treatment was administered. HU treatment, at both 5mM and
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Figure 6 Increased i-cell derivatives migration towards the foot following HU treatment.

Hydra vulgaris head (A) and foot (B) after transplantation of transgenic graft tissue onto non-treated control host showing an increased
number of migrated fluorescent cells in the head compared to the foot. Unchanged number of fluorescent cells in the head of Hydra
vulgaris after 10mM HU (C) and 5mM HU (E) treatment. Increased number of fluorescent cells in the foot of Hydra vulgaris after 10mM
HU (D) and 5mM HU (F) treatment.

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