Investigating the basis of animal regeneration
with the Hydra model system
Dominik Zürcher, Kantonsschule Büelrain Winterthur
Supervision: S. Al-Haddad, Y. Wenger, Genetics and Evolution , Unige
Introduction
Hydra is a simple model organism. It belongs to the phylum Cnidaria, the subphylum Medusozoa and the class
Hydrozoa. Hydra can be found in unpolluted fresh water ponds, lakes and streams. They are carnivores; they
eat Crustaceans like Daphnia and Artemia. Biologists are especially interested in the high regeneration
potential of Hydra.
Hydra has a simple morphology represented by a tubular body. On the basal end,
a basal disk allows it to fix to a substrate. On the apical side, it has a mouth
surrounded by tentacles to capture the prey. Hydra has two body layers: the
outer layer is the ectoderm and the inner layer is the endoderm. The two layers
are separated by the mesoglea. The length of a Hydra is between 0.5 and 1cm.
Figure 1. Morphology and
Anatomy of Hydra.
Figure 2. Reproduction mode in Hydra
When Hydra has enough food and is in a comfortable environment it reproduces asexually by growing small
buds in the budding zone. If the conditions are hard (starvation, very low temperature), Hydra reproduces
sexually. It can turn into a male by developing testis on the upper part of the body column, or it turns into a
female and develop ovaries on the lower part of the body.
After a bisection, Hydra starts to
regenerate. In the upper part,
a foot starts to grow; this is called
foot regeneration (FR). From the
lower part, a head formation is
observed; this is the head
regeneration (HR). Many proteins
Figure 3. Regeneration process in Hydra
regulate the HR and the FR. One
of the HR regulators is CREB; it plays an important role in the MAPK/CREB pathway. When the activity of CREB
is blocked, Hydra do not regenerate anymore.
Many proteins are used as markers for head formation like RFamide. It is a neuropeptide found in nerve cells
forming a net in the head of an intact Hydra. During the head regeneration process, the nerve net system
reforms and can be visualized by using an antibody to detect the RFamide.
The project addressed 2 main questions: the first one is to see if CREB is conserved in other organisms, and the
second one is to compare the head regeneration process between decapitation and mid gastric bisection by
following the formation of the nerve net in the head.
Methods
Hydra Fixation
I put 15 Hydra (endo GFP+) for each condition into 2 ml tubes with 500 μl of hydra medium, then I added 500μl
of 4% Urethane (diluted in Hydra medium) to let Hydra relax and elongate. After this, I washed the Hydra with
a fixative solution called Lavdovsky minus and incubated them in this solution for 1h at 37°C. Then, I washed
them with Ethanol (EtOH) 100%.
Lavdovsky minus
EtOH 100 %
Formaldehyde
H20
50.0%
3.7%
46.3%
Immuno-Fluorescence
I started a gradual rehydration with EtOH 70%, EtOH 50% and EtOH 30% in PBS1x for 5min at RT for each
wash. The next step was a permeabilization to help the antibody to penetrate into the cells. For that, I added
0.5%Triton X-100 and left the tubes for 15 minutes at RT. Then, Hydra were washed 3 times with PBS1x for 5
minutes each. A blocking solution 2%BSA in PBS (Bovine serum albumin) was added for 1 hour at RT. I added
the primary antibody anti-RFamide, diluted to 1/1000 in BPS 2%, to each tube and let them incubate ON at 4°C.
On the next day, I washed all the tubes 3 times with PBS1x.
From now on everything had to be kept in the dark for not to bleach the fluorescent signal: I added 100μl of the
secondary antibody anti-rabbit diluted to 1/400 in PBS and incubated my animals for 4h at RT. I washed them
3 times again with PBS1x, and I added 500μl of Dapi (diluted at 1/5000 in PBS1x) for 10min at RT. After
washing 2 times with PBS1x, I washed them briefly with H2O.
Imaging
To observe the fluorescent signal under the microscope, I mounted my Hydra on slides. To stick the cover slip
on the slide, I used 15 μl of Mowiol and left them to dry till the next day, in the dark.
The imaging was done under a fluorescent microscope with the same picture settings for each filter (red for
RFamide and blue for Dapi).
Phylogenetic analysis
After obtaining the CREB sequence for Hydra, I did a blast with the blast tool using the NCBI site to search for
similar CREB sequences for the following organisms: human, mouse, xenopus (frog), chicken, zebrafish,
drosophila (fruit fly) and amphimedon (sponge).
All the sequences were aligned together using the T-coffee alignment software, and the tree was done using
PhyML.
Results
Phylogenetic analysis of CREB sequence in Hydra
Because of the important role of CREB in regeneration, it is interesting to see if CREB is conserved in other
model organisms.
A
B
Figure 4. Phylogenetic analysis to study the conservation of CREB domains through evolution.
A. Alignment of CREB sequences from different model organisms; two conserved domains pKID and bZIP are
shown. B. Phylogenetic tree with PhyML showing a high bootstrap value of 96 at the base of tree.
Follow the Head Regeneration process in Hydra
Transgenic Hydra expressing the GFP in endodermal epithelial cells were cut at two different levels: midgastric and decapitation (just below the head). Every day, Hydra were pictured to follow the head formation
and to compare the regeneration speed between the two cuts.
Figure 5. Hydra cut at the mid-gastric level and decapitation
These Hydra were pictured with bright field and fluorescent microscope. The
fluorescence comes from the GFP signal in transgenic Hydra.
The animals were followed day by day to study the regeneration process. (T0=0h,
T1=24h, T2=48h, T3=72h).
Detection of RFamide in intact and regeneration conditions
RFamide plays the role of a marker for nerve cells that localize in the head region. We wanted to compare the
re-formation of the nerve cells after 24h of head regeneration in mid-gastric and decapitation conditions.
Figure 6. Immunofluorescence for RFamide detection on intact animals.
After fixation, the animals were immuno-stained with anti-RFamide. The first panel
shows the RFamide signal in red, the second panel shows the nuclear staining Dapi in
blue and the third panel shows the merge between the red and blue.
Figure 7. Immunofluorescence for RFamide detection after a mid-gastric bisection.
These animals were cut at the mid gastric level and were fixed right after (T0) or after 24h
(T24). The first panel shows the RFamide signal in red, the second panel shows the
nuclear staining Dapi in blue and the third panel shows the merge between the red and
the blue.
Figure 8. Immunofluorescence for RFamide detection after decapitation.
These animals were decapitated then fixed right after cutting (T0) or after 24h (T24).
The first panel shows the RFamide signal in red, the second panel shows the nuclear
staining Dapi in blue and the third panel shows the merge between the red and the blue.
Discussion and Conclusion
After the CREB phylogenetic analysis, I could see the high value of bootstrap (96) which indicates a very high
support for the model of the tree: the Hydra CREB has orthologs in other model organisms and is well
conserved specially at the level of two domains pKID and bZIP as we can see in the protein alignment.
CREB is very important for the regeneration process in Hydra especially for the HR. Since it is conserved in
other species, perhaps it can play a regeneration role in organisms that have a low regenerative capacity.
During the process of live imaging, I could observe how Hydra regenerate after bisection. Between 1 and 2 days
after bisection, the tentacles start to grow in both conditions. After 3 days, the tentacles from the decapitation
condition are almost fully regenerated whereas the tentacles of the mid-gastric bisection are in an earlier stage.
After I took the pictures of the immunofluorescence, I could see that nerve net formation in HR after a midgastric bisection is faster then after a decapitation. Right after the mid-gastric bisection no nerve cells are
observed, whereas some nerve cells remain present next to the head region after decapitation. After 24 hours,
the nerve net is already highly regenerated in both conditions. The nerve net formation in the decapitation
condition is not as developed as the one in the mid-gastric condition. The mid-gastric is almost fully
regenerated. I think it is the case because, the smaller the pieces are, the faster they regenerate.
Hydra is an ideal animal to investigate the regeneration process. Even if it is cut at different levels, it can still
regenerate. The mechanism behind head regeneration in mid-gastric and decapitation is probably not the same,
but this should be more investigated. Since CREB is conserved in other organisms, it would be interesting to try
to push its activity in animals that do not regenerate well, and see if the regeneration capacity can be improved.
Acknowledgement
I would like to thank the team of the laboratory of B. Galliot for giving me the chance to work there. A special
thanks go to Sarah al Haddad and Yvan Wenger who mentored me during the week. They both did a great job
and explained me everything in a very good way.
I would also like to thank Swiss Youth at Science. Without them I wouldn’t have the chance to participate in this
week of study. It was a great experience for me to see a laboratory for the first time.