admin
Fri, 02/09/2024 - 19:51
Edited Text
“Investigation Into the Effects of Temperature Stress on Caudal Fin Regeneration in
Zebrafish”
An Honors Thesis
by
Eric Moeller
California, Pennsylvania
2019
Contents
1 Introduction
1
1.1
An overview of the fin regeneration process . . . . . . . . . . . . . . . . . . .
2
1.2
Molecular mechanisms underlying regeneration . . . . . . . . . . . . . . . . .
3
1.3
The effects of temperature on regeneration . . . . . . . . . . . . . . . . . . .
5
1.4
Heat-shock proteins and stress responses . . . . . . . . . . . . . . . . . . . .
6
2 Methods
7
2.1
Zebrafish care and maintenance . . . . . . . . . . . . . . . . . . . . . . . . .
7
2.2
Anaesthesia and amputation of the caudal fin
. . . . . . . . . . . . . . . . .
7
2.3
Tissue staining for analysis of fin branching . . . . . . . . . . . . . . . . . .
7
2.4
Immunohistochemistry to analyze differences in signaling proteins expressed
2.5
in regenerating fins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8
RNA analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8
3 Results
8
3.1
Ray branching . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8
3.2
Immunohistochemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
12
3.3
RNA analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
14
4 Discussion
15
California University of Pennsylvania
1
Eric Moeller
Introduction
The ability to regrow amputated limbs holds much interest in the field of biology. The
process of regeneration reactivates pathways that are used during initial limb development
as an embryo, which are conserved in humans. Research into regeneration may discover new
treatments that enhance human wound healing, possibly even allowing for the regrowth of
limbs. Regeneration follows a unique and complex process that is not often observed outside
of teleost fish and urodele amphibians. Zebrafish, for example, have a remarkable ability to
regenerate acutely damaged fins (Johnson and Weston, 1995), optic nerves (Goldman et al,
2001), and parts of their heart (Poss et al, 2000). This ability was first observed in 1786 by
the French scientist Broussonet who found that when zebrafish lost part of their caudal fin,
they were able to regrow them within 2-4 weeks (Pfefferli and Jazwinska, 2015).
In mammals, fibrosis is the dominant reaction to substantial injuries. This results in
the formation of scar tissue, which can cause discomfort and loss of function. In zebrafish,
however, this initial fibrin deposition is broken down to allow for regeneration. If regeneration
is inhibited, fibrin is not broken down and the healing ceases at scar formation. It has been
proposed that scarring precedes regeneration, and the proliferation of myocytes determines
whether wound healing will cease at scar formation or lead to regeneration (Poss et al, 2002).
Research into zebrafish regeneration allows scientists to more fully understand the mechanistic pathways involved in tissue regeneration. This understanding could have impacts
in many scientific fields through expanding our knowledge of genetics and cell signalling
(Gemberling et al, 2013). Novel discoveries could also aid in the development of medical
treatments if regeneration becomes understood fully enough to induce wound healing to
follow similar pathways in humans.
Page 1 of 22
California University of Pennsylvania
1.1
Eric Moeller
An overview of the fin regeneration process
The fin of teleost fish is mainly comprised of bony rays that support the structure of the
fin, inter-ray mesenchymal cells, and epidermal cells. Blood vessels and nerve axons extend
through both the rays and the mesenchymal tissue. The bony rays are maintained and created by osteoblasts, which function to deposit bone along the rays. The caudal fin fold is
stabilized by 16-18 bony rays that occassionally branch named lepidotrichia. The distal ends
of the rays, called actinotrichia, remain uncalcified while the rest of the structure is mineralized (Pfefferli and Jazwinska, 2015). In zebrafish, characteristic stripes of chromatophores
extend the length of the body, resulting in a dark striped pattern.
When the fin is amputated, regeneration begins quickly. Within a few hours, cells cover
the wound to protect it, forming a structure called the apical ectodermal cap which plays a
similar role to the apical ectodermal ridge in limb development. Within 1-2 days of wound
cap formation, dedifferentiated cells migrate underneath the epidermal wound cap and form
a white opaque mass called a blastema (Gemberling et al, 2012). For example, mature
osteoblasts have been found to dedifferentiate and migrate to form part of the blastema.
These cells are lineage-restricted, which has also been observed in amphibians. (Pfefferli
and Jazwinska, 2015; Quint et al, 2002). Blastema formation distinguishes regeneration
from limb development, however the rest of the outgrowth of the fin follows developmental
processes (Manuel and Carmen, 2010).
The blastema is formed from at least two lineages, osteoblasts and fibroblast-like cells
(Knopf et al, 2011). Cells within the blastema form two distinct groups known as a distal
blastema and a proximal blastema. The distal blastema proliferates slowly, providing a direction for the outgrowth and contributing daughter cells to the proximal blastema. The
proximal blastema proliferates rapidly and cells within it redifferentiate to produce the structures that were lost (Iovine, 2007).The cells of the blastema rapidly begin to migrate distally
from the site of amputation, and maintain a milky white appearance for the first few days
of tissue regeneration. This process continues, proceeding distally until the fin is reformed.
Page 2 of 22
California University of Pennsylvania
Eric Moeller
The regeneration process takes around 2-4 weeks depending on water conditions, and is not
affected by repeated amputations (Azevedo et al. 2011).
Figure 1: Diagram of the fin regeneration process (Pfefferli and Jazwinska, 2015)
1.2
Molecular mechanisms underlying regeneration
It has been proposed that the distal blastema acts as a signaling center during the regeneration process through the Wnt signaling pathway, which regulates epidermal patterning,
cell proliferation, and osteoblast differentiation through fibroblast growth factor (FGF) and
bone morphogenic protein (BMP) pathways (Wehner et al, 2014). Redifferentiation of the
osteoblasts in the proximal blastema requires a downregulation of the Wnt pathway by BMP
signaling, so the interplay between these two signaling pathways determines the dedifferentiation and redifferentiation of osteoblasts (Stewart et al, 2014).In development, FGF genes
function in wound healing, mesoderm induction, mature tissues/systems angiogenesis, and
the proliferation of fibroblasts, which fills up the wound space/cavity early in the wound healing process. FGF is a small secreted molecule that activates downstream signaling events in
nearby cells by binding to it’s cognate receptor, the FGF receptor. There are several FGF
genes and FGF receptors. FGF is expressed in mesenchymal cells underlying the wound
epidermis during blastema formation and in distal blastemal tissue during regenerative outgrowth (Poss et al. 2000).
Cells exppressing Fgfr1 create a signaling center that is required for the creation of the
proximal and distal blatema (Poss et al, 2002; Poss et al, 2000; Nechiporuk and Keating,
Page 3 of 22
California University of Pennsylvania
Eric Moeller
2002). FGF signaling in the distal blastema regulates cell proliferation in the proximal
blastema, while in the lateral basal epidermal layer it is required for bone differentiation and
is represented by the expression of Shh (Iovine, 2007). Shh signaling controls the patterning
of the regenerating tissue in a concentration-dependent manner. Shh active cells also secretes
Gre, which suppresses BMP signaling. BMP in turn inhibits FGF signalling, causing the
cells to stop growing (Selever et al, 2004). Shh and bmp2b can stimulate the secretion of
bone matrix within the blastema, possibly by influencing the differentiation of osteoblasts.
When Shh and FGF are close in proximity to each other, Shh inhibits the downregulating
BMP signal via Gre, allowing FGF to be expressed and growth to occur. As the tissues
move further apart, the Gre signal becomes less concentrated, and at a certain concentration
threshold the BMP signal becomes strong enough to inhibit FGF, causing the tissue to stop
growing (Freeman, 2000).
Figure 2: Diagram of pathways involved in limb formation (Freeman, 2000)
During fin regeneraion, Shh has been shown to be upregulated in the lateral basal epidermal layer around the area where the fin rays are regenerating. Other genes controlled
by Shh are induced in the basal epidermal layer and in nearby mesenchymal cells (Smith et
al, 2006). Expresison of shh or bmp2 between fin rays leads to upregulation of osteoblast
transcription factors which results in the formation of bone. This suggests that either Shh
Page 4 of 22
California University of Pennsylvania
Eric Moeller
or BMP is sufficient for bone formation (Quint et al, 2002). Knockout of Shh alone does
not result in a lack of bone formation, however chordin inhibition of BMP’s prevents the
expression of genes involved in the differentiation of osteoblasts, the development of mature
osteoblasts, and mineralization of the fin rays, which suggests that BMP is required for ray
development (Smith et al, 2006).
Both Sonic hedgehog and Indian hedgehog are activated in the blastemal tissue and
regulate blastemal proliferation, maintenance, and tissue growth (Iovine 2007). Interactions
with other signaling pathways including FGF and Wnt signaling has been shown to amplify
the regenerative response during fin regeneration (Lee et al. 2005). FGF activates and
regulates blastemal proliferation, causing extension of the tissue. It also controls epidermal
gene expression and maintains the expression of WNT signaling (wntb5), proximal Shh,
and lef1. It has been suggested that FGF may stimulate blastemal outgrowth through the
maintenance of Shh signaling, which is similar to their roles in limb development (Pfefferli,
2015).
1.3
The effects of temperature on regeneration
Zebrafish live in areas with temperatures that fluctuate often, and as such they can tolerate
a range of temperatures without lasting harm to the fish. In their natural habitat of north
east India, Bangladesh and Nepal, the lowest water temperature recorded was 14.2 ◦ C and
the highest was 33 ◦ C (Lopez-Olmeda and Sanchez-Vazquez, 2011). temperature stress is
only one form of stress which may have negative impacts on wound healing. In general,
stress is defined as a condition that disturbs the normal function of the biological system
or a condition that decreases fitness (Hoffmann and Parsons, 1991; Bijlsma and Loeschcke,
1997). If temperature stress affects wound healing, other sources of stress may also have
negative effects on wound healing in humans.
Page 5 of 22
California University of Pennsylvania
1.4
Eric Moeller
Heat-shock proteins and stress responses
Heat shock proteins are a class of chaperonin proteins that help proteins within a cell to fold
correctly. As the polypeptide chains are being transcribed, chaperonin proteins guide the
folding process. Apart from this function, molecular chaperones are involved in transport,
folding, unfolding, assembly, and disassembly of multi-structured units and degredation of
misfolded or aggregated proteins (Sorensen, Kristensen, and Loeschcke 2003). These proteins
also play a role in coping with oxidative and temperature stress, both of which cause proteins
to partially denature. If proteins refold incorrectly, they can become nonfunctional and
potentially harmful to the cell. To prevent this from occurring, chaperonin proteins also
work to guide the refolding process (Kim et al. 2013). When tissue is regenerating, protein
production is increased due to the need for cells to grow and proliferate quickly, resulting
in an increased demand for chaperonin proteins. Temperaure stress would also cause an
increase in demand for caperonin proteins, and it has been previously shown that when
there is a lack of chaperones, errors occur (Chaudhuri and Paul, 2006).
We hypothesize that fins regenerating in higher temperatureswould show errors in regeneration, and that the errors could be caused by an increased demand for heat-shock proteins
during temperature stress. Under temperature stress the chaperonin proteins are dealing
with the stress of the warmer temperatures, so more errors in protein folding could occur
within the rapidly dividing cells within the blastema. If developmental proteins involved in
cell to cell signalling are functioning abnormally, less signal would be received by cells and
they would not develop the overall tissue as precisely. Errors in cell signalling could cause
less organization in the overall tissue and lower protein expression of developmental signaling proteins within the tissue. If temperature stress increases the workload of chaperonin
proteins, there should be a significant upregulation of chaperonin gene expression in both
cold and warmer temperatures when compared to normal temperature subjects.
Objectives
• To determine if temperature stress affects regenerated fin morphology by examining
Page 6 of 22
California University of Pennsylvania
Eric Moeller
differences in fin branching under different temperature conditions
• To determine whether there are differences in the levels of key signaling signals used
during fin regeneration under normal conditions and under temperature stress
2
2.1
Methods
Zebrafish care and maintenance
Three groups of 5-15 fish were kept in 10 gallon tanks, each with different water temperatures
(22◦ C, 28◦ C, 34◦ C). The three tanks were maintained under the same light/dark conditions
and they had the same feeding regimine (1-2 times per day).
2.2
Anaesthesia and amputation of the caudal fin
Fish were caught and submersed in a 0.16% solution of Tricane (ms-222) until muscle activity
ceased, then were transferred to a sterile petri dish. Approximately half of the fin was amputated using a sterile razor, after which the fish were transferred to a recovery beaker until
they began to swim normally. Once swimming appeared normal, the fish were transferred
back to the appropriate tanks.
2.3
Tissue staining for analysis of fin branching
Fin segments were collected at 0dpa, 7dpa, or 14dpa and rinsed in phosphate buffered saline
(PBS). The segments were then placed in 95% ethanol on ice for1hr before being transferred
to fresh 95% ethanol at room temperature overnight. Bone and Cartilage stain solution of
0.02% alcian blue and 0.005% alizarin red was added and left overnight at room temperature
with rocking. The fins were rinsed in a series of glycerol/0.25% KOH solutions and stored
in 50% glycerol/0.25% KOH for further clearing and storage. Fins were mounted in glycerol
and ray branching data was collected with a compound microscope.
Page 7 of 22
California University of Pennsylvania
2.4
Eric Moeller
Immunohistochemistry to analyze differences in signaling
proteins expressed in regenerating fins
Fins were collected after 4dpa and fixed in 4% paraformaldehyde at 4◦ C overnight. The
fins were rinsed five times with phosphate buffered saline with 1% tween20 (PBS-tween),
incubated in normal horse serum (30 µl), then the primary antibody dilution was added
and incubated at 4◦ C overnight. Fins were treated with antibodies for Gre (1:500), Shh
(1:500), HSP-70, FGF4 (1:500) or ZNS-5 (1:200). Fins from all three temperature treatments
were labeled with ZNS-5, hedgehog, Gremlin and FGF. Fins from the normal and warm
temperature treatments were labeled with HSP-70. The fins were rinsed four times PBStween, then a biotinylated second antibody dilution was added (anti-mouse 1:200). The
biotinylated antibody was detected using a vecta-stain ABC kit. Staining was imaged using
a compound microscope.
2.5
RNA analysis
RNA was isolated using TRIzol (Invitrogen) or RNeasy (Qiagen). Twelve fins were cut
from each temperature and samples from the same temperature were pooled to increase the
amount of RNA. Absorbtion was measured to measure RNA levels. A Superscript III firststrand synthesis kit (Invitrogen) was used to convert the RNA into complimentary DNA
(cDNA). PCR was performed to amplify FGF3 transcripts, and the results were analyzed
using agarose gel electrophoresis.
3
3.1
Results
Ray branching
Fin ray branching in regenerated fins was examined after 14 days (14 dpa), in fins taken
from fish regenerating in cold, normal and warm temperatures. A one-way ANOVA showed
Page 8 of 22
California University of Pennsylvania
Eric Moeller
that the degree of fin ray branching seen after regeneration was significantly different in
cold and warm temperatures, compared to normal temperatures (F2,614=668.33; P¡0.0001).
Post hoc comparisons using the Tukey HSD test indicated a lower degree of ray branching in
regenerated fins in cold temperature conditions (M = 0.477, SD = 0.501) compared to fish
regenerating in normal temperature conditions (M = 1.054, SD = 0.424) at 14dpa. There
were also significant differences in ray branching between regenerated fins in the cold and
warm temperature conditions (M = 1.928, SD = 0.424), as well as a difference in branching
between regenerated fins under normal and warm temperature condition (Cold vs. Normal
P ¡ 0.01, Cold vs. Warm P ¡ 0.01, Normal vs. Warm P ¡ 0.01).
We also compared how the amount of ray branching had changed over time, by comparing
ray branching between 0dpa and 14dpa fish for the cold, normal, and warm conditions,
and found that there are significant differences in each group. Fins regenerating in cold
temperature conditions had a lower degree of branching at 14dpa (M = 0.477, SD = 0.500)
compared to fins at 0dpa in cold conditions (M = 1.689, SD = 0.619) (t = 21.01; df =
335.01; P ¡ 0.0001) (Figure 3). Fins regenerating in the warm temperature conditions has a
higher degree of branching at 14dpa (M = 1.928, SD = 0.423) compared to fins at 0dpa in
the warm conditions (M = 1.736, SD = 0.543) (t = -3.61; df = 234.2; P ¡ 0.001) (Figure 4).
The fins regenerating in the normal temperature conditions had a lower degree of branching
at 14dpa (M = 1.054, SD = 0.323) compared to fins in the normal temperature conditions
at 0dpa (M = 1.75, SD = 0.534) (t = 13.41; df = 233.98; P ¡ 0.0001) (Figure 5).
Page 9 of 22
California University of Pennsylvania
Eric Moeller
Figure 3: The degree of branching observed after 0dpa and 14dpa post-regeneration in
fins regenerating in the cold (21◦ C) conditions.
Figure 4: The degree of branching observed at both 0dpa and 14dpa post-regeneration in
fins regenerating in normal (28◦ C) conditions.
Page 10 of 22
California University of Pennsylvania
Eric Moeller
Figure 5: The degree of branching observed at both 0dpa and 14dpa post-regeneration in
fins regenerating in warm (34◦ C) conditions.
Figure 6: The percent change in branching before and after regeneration in cold, normal,
and warm temperatures. A positive percentage indicates that the number of fins with that
degree of branching increased post-regeneration, while a negative percentage indicates that
the number of fins with that degree of branching decreased post-regeneration.
Page 11 of 22
California University of Pennsylvania
3.2
Eric Moeller
Immunohistochemistry
1. FGF expression
The fin regenerating in the cold temperature conditions (a) showed a very low expression of FGF (shown in purple) compared to the fin regenerating in the normal
conditions (b). The fin regenerating in the warm conditions (c) showed a greater
amount of FGF expression than the fin regenerating in cold conditions, but showed
less expression compared to the fin regenerating in the normal condition.
(a) Cold 22◦ C
(b) Normal 28◦ C
(c) Warm 34◦ C
Figure 7: FGF expression in zebrafish caudal fins at 4dpa post-regeneration
2. ZNS-5 osteoblast expression
More osteoblasts were observed during regeneration at elevated temperatures (b) compared to the normal temperaures (a) at 4dpa (ZNS-5 shown in purple). No osteoblasts
were seen in the cold 4dpa regenerated fins (not shown).
(b) Normal 28◦ C
(c) Warm 34◦ C
Page 12 of 22
California University of Pennsylvania
Eric Moeller
Figure 8: ZNS-5 expression in zebrafish caudal fins at 4dpa post-regeneration. The
arrow indicates a labeled osteoblast
3. HSP-70 expression
A higher degree of staining was seen in the fins that regenerated in the warm temperature (b) compared to the normal temperature (a).
(a) Normal 28◦ C
(b) Warm 34◦ C
Figure 9: HSP-70 expression in zebrafish caudal fins at 4dpa post-regeneration.
4. Gremlin expression
In the cold 4dpa regenerated fins, the gremlin expression was low and seemed mainly
concentrated at the distal edge of the blastema. The normal 4dpa regenerated fins had
a similar amount of expression, however the majority of the Gre expression appeared
to be concentrated at the site of amputation as well as at tip of the blastema. The
warm 4dpa regenerated fins had a higher expression at the site of amputation as well
as at the tips of the fin folds in the blastema.
Page 13 of 22
California University of Pennsylvania
(a) Cold 22◦ C
Eric Moeller
(b) Normal 28◦ C
(c) Warm 34◦ C
Figure 10: Gremlin expression in zebrafish caudal fins at 4dpa post-regeneration.
5. Sonic hedgehog expression
Hedgehog expression appeared to be concentrated at the site of amputation as well as
along the apical edge of the blastema in the cold and normal 4dpa fins. The warm
4dpa fins had a lower expression along the site of amputation, but appeared to have a
greater expression at the fin tips.
(a) Cold 22◦ C
(b) Normal 28◦ C
(c) Warm 34◦ C
Figure 11: Sonic hedgehog expression in zebrafish caudal fins at 4dpa
post-regeneration.
3.3
RNA analysis
No FGF3 was seen in the negative control or in RNA extracted from fins regenerating in cold
conditions. Strong bands were seen in RNA from both the normal and the warm conditions,
Page 14 of 22
California University of Pennsylvania
Eric Moeller
as seen in figure 8.
Figure 12: Agarose gel electrophoresis of FGF3 RNA transcripts extracted from fish in
cold (22◦ C), normal (28◦ C), and warm (34◦ C) conditions.
4
Discussion
In investigating whether heat stress affects fin morphology after regeneration, we found
that there are differences in branching, protein expression, and visual characteristics. After
Page 15 of 22
California University of Pennsylvania
Eric Moeller
regenerating in the warm conditions, there is a greater amount of ray branching compared to
fins that regenerate under normal temperature conditions. This suggests that fin regeneration
is affected by temperature stress. In the cold conditions, we saw a lower amount of ray
branching compared to fins that regenerated under normal temperature conditions. We
believe this indicates a slower regeneration rate at cold temperatures rather than abnormal
fin regeneration. This temperature dependent rate decrease has been observed before the fin
rays that regenerated in the normal conditions also had a reduction in branching compared
to initial branching, which might indicate that the fins have not fully regenerated at 14dpa.
The regeneration process is typically completed after approximately three weeks, so the
process may still be occurring at 14dpa (Pfefferli and Jazwinska, 2015).
Along with these differences in branching, other morphological abnormalities were seen.
The rays appeared to have a rougher appearance and appeared thicker in the cold and warm
conditions compared to the normal conditions. The bands of melanophores which give the
zebrafish their characteristic striped appearance was darker in the warm conditions compared
to those of fish kept in the cold conditions. This is consistent with previous findings, which
show that the number of melanophores decreased under cold temperature conditions of 17◦ C
compared to zebrafish in normal temperature conditions of 26.5◦ C (Kulkeaw et al., 2011).
Fish in the warm tank were also more energetic than the fish in the cold conditions, however
they appeared to succumb to the effects of the tricaine in a much shorter time frame. These
observations further suggest that temperature stress is having an effect on regeneration and
could be an interesting avenue to pursue in the future.
In addition to ray organization, there appeared to be differences in bone ray thickness
between fish regenerating in normal and warm temperatures. To investigate these differences,
osteoblasts were observed. Cell signaling networks direct osteoblast progenitors to rebuild the
fin rays, so an increase in osteoblasts may cause an increase in bone deposition (Armstrong et
al., 2017). There appeared to be a greater number of osteoblasts in the warm temperatures
compared to fins in the normal temperatures (Figure 9). This may explain why the rays
Page 16 of 22
California University of Pennsylvania
Eric Moeller
were thicker and less organized. If there are more osteoblasts producing bone than normal, a
greater amount of bone deposition would occur, causing a thickening of the rays. Osteoblasts
were not observed in the cold-regenerating fins at 4dpa, again likely due to the delay in
regeneration at this temperature.
Given that differences in ray branching were observed, we wanted to determine the molecular basis of these abnormalities. To examine this, we observed the expression of signaling
molecules within the regenerating fins at 4dpa, early in the regrowth process. We first looked
at the expression of FGF, a protein that is thought to be a key signaling factor within the
blastema. FGF is expressed in mesenchymal cells underlying the wound epidermis during
blastemal formation and in the distal blastemal tissue during the regeneration process (Poss
et al. 2000). Because FGF is secreted from the tip of the blastema in a gradient, we expected to see the greatest amount of FGF expression at the tip of the fin, with a gradual
lessening of expression further away from the blastema. This expression pattern can be seen
in the fin regenerating in normal temperature conditions (Figure 8b) but cannot be seen in
fins regenerating in cold conditions at 4dpa (Figure 8a). The fin regenerating in the cold
condition did not show an appreciable amount of FGF expression, and the physical shape of
the fin tip was flat whereas the fin tips in the normal and warm had distinct ridges. Again,
this may have been due to the slower rate of regeneration under cold temperature conditions.
This could be confirmed by looking at FGF expression later in the regeneration process.
The fin in the warm condition did show FGF expression, however the FGF was not
expressed in the same pattern as in the normal temperature (Figure 8c). The expression
appeared patchy, with some areas of high concentration at the tip of the blastema and
some areas with no FGF expression. This suggests that temperature stress affects levels
of signaling molecules during regeneration, supporting our hypothesis. The differences in
the expression pattern of FGF could explain the differences in morphology that we saw
in fins regenerating under temperature stress. FGF regulates cell proliferation and bone
differentiation, so differences in FGF signaling could cause morphological differences in the
Page 17 of 22
California University of Pennsylvania
Eric Moeller
regenerated fin (Iovine, 2007). To confirm that the difference in FGF levels are not the
result of reduced gene expression, but are rather specifically due to reduced protein levels,
indicative of issues with protein folding, we examined levels of FGF transcripts. We saw
similar levels of FGF3 RNA in both the normal and warm conditions, however no expression
was observed in the cold condition or in the negative control. The RNA bands for FGF
in the normal and warm conditions appeared to be the same strength, indicating that the
fins in both conditions are producing the same level of RNA to create FGF3 proteins. This
indicates that gene expression is not affected by temperature at the level of transcription and
supports the idea that functional protein levels are being affected by temperature stress. If
the protein expression of FGF is lower in fins regenerating in warm temperatures compared
to normal temperatures, then the difference was occurring on the protein level and is likely
due to the lack of availability of chaperone proteins (heat-shock proteins) that normally
help ensure proteins are folded correctly. Temperature stress has been shown to cause an
increased demand for chaperonin proteins, and errors occur when there is an insufficient
supply to deal with the stress (Chaudhuri and Paul, 2006). The lack of RNA and protein
expression for FGF in the cold condition is likely due to the slower regeneration time under
lower temperatures rather than temperature stress.
To confirm that the fish were under temperature stress, we labeled a heat shock protein
that is known to aid cellular processes under temperature stress (HSP-70). We saw a greater
expression of HSP-70 in the elevated temperature conditions compared to the normal conditions (Figure 9), which indicated that fish in the elevated temperature were experiencing
a greater amount of stress.
The expression of another signaling protein, gremlin, was investigated. In development
this molecule acts as a concentration dependent inhibitor of BMP, which in turn is a concentration dependent inhibitor of FGF. As the Shh and FGF signaling centers move further
apart, gremlin becomes less concentrated. When gremlin concentration drops to a certain
point, BMP is no longer inhibited and can suppress FGF, causing the tissue to stop growing
Page 18 of 22
California University of Pennsylvania
Eric Moeller
(Freeman, 2000). We found that the expression of gremlin was lower in both the cold and
warm conditions compared to normal (figure 9a, c). A lower expression of gremlin may allow
BMP to inhibit FGF, which could cause the differences in FGF expression that is seen in
the cold and warm conditions. Sonic hedgehog expression appeared to be concentrated at
the site of amputation and at the tip of the fins in the cold and normal fins, with a lower
expression in the center of the regenerating fins. In the warm 4dpa fin, Shh expression
was greatest along the tip of the blastema and appeared to be greater throughout the fin
compared to the fins regenerating in normal and cold temperatures. Shh signaling regulates
blastemal proliferation and tissue growth (Iovine, 2007), so a greater amount of Shh expression in the warm temperatures could lead to differences during the regeneration process and
may explain the morphological differences that were seen.
If the amount of heat stress proteins were insufficient to deal with the combined stress of
increased temperature as well as the large increase in cell division and growth, some proteins
will become nonfunctional. Our results appear to support this, as there was a lower level of
FGF protein expression in the warm conditions without a difference in the amount of RNA
transcripts for FGF3. These results, along with the other differences observed, indicate that
temperature stress does cause morphological differences in regeneration.
References
[1] Armstrong, B.E., Henner, A., Stewart, S., and Stankunas, K. (2017). Shh promotes
direct interactions between epidermal cells and osteoblast progenitors to shape regenerated
zebrafish bone. Development 144, 1165-1176
[2] Azevedo, A.S., Grotek, B., Jacinto, A., Weidinger, G., Saude, L. (2011). The Regenerative
Capacity of the Zebrafish Caudal Fin Is Not Affected by Repeated Amputations. PLoS
ONE 6(7), 1-8.
Page 19 of 22
California University of Pennsylvania
Eric Moeller
[3] Bjilsma, R. and Loeschcke, V. (1997). Environmental Stress, Adaptation and evolution.
Birkhauser Verlag, Basel.
[4] Chaudhuri, T.K., and Paul, S. (2006). Protein-misfolding diseases and chaperone-based
therapeutic approaches. The FEBS Journal 273(7). 1331-1349.
[5] Matthew, F. (2000). Feedback control of intercellular signalling in development. Nature
408, 313-319.
[6] Gemberling, M., Bailey, T.J., Hyde, D.R., Poss, K.D. (2013). The zebrafish as a model
for complex tissue regeneration. Trends in Genetics 29, 611-620.
[7] Goldman, D., Hankin, M., Li, Z., Dai, X., Ding, J. (2001). Transgenic zebrafish for
studying nervous system development and regeneration. Transgenic Research 10, 21-33.
[8] Hoffman, A.A., and Parsons, P.A. (1991). Evolutionary Genetics and Environmental
Stress. Oxford University Press 4, 248
[9] Iovine, M.K. (2007). Conserved mechanisms regulate outgrowth in zebrafish fins. Nature
Chemical Biology 3, 613-618.
[10] Johnson, S.L., Weston, J.A. (1995). Temperature-Sensitive Mutations That Cause StageSpecific Defects in Zebrafish Fin Regeneration. Genetics 141(4), 1583-1595.
[11] Kim, Y.E., Hipp, M.S., Hayer-Hartl, A.B.M., and Hartl, U.F. (2013). Molecular Chaperone Functions in Protein Folding and Proteostasis. Annual Review of Biochemistry 82,
323-355.
[12] Knopf, F., Hammond, C., Chekuru, A., Kurth, T., Hans, S., Weber, C.W., Mahatma,
G., Fisher, S., Brand, M., Schilte-Merker, S., Weidinger, G. (2011). Bone regenerates via
dedifferentiation of osteoblasts in the zebrafish fin. Developmental Cell 20, 713-724.
Page 20 of 22
California University of Pennsylvania
Eric Moeller
[13] Kulkeaw, D., Ishitani, T., Kanemaru, T., Ivanovski, O., Nakagawa, M., Mizuochi, C.,
Horio, Y., and Sugiyama, D. (2011). Cold exposure down-regulates zebrafish pigmentation.
Genes to Cells 16, 358-367.
[14] Laforest, L., Brown, C.W., Poleo, G., Géraudie, J., Tada, M., Ekker, M., Akimenko,
M.A. (1998). Involvement of the sonic hedgehog, patched 1 and bmp2 genes in patterning
of the zebrafish dermal fin rays. Development 125(21), 4175-4184.
[15] Lee, T., Grill, S., Sanchez, A., Murphy-Ryan, M., Poss, K.D. (2005). Fgf signaling
instructs position-dependent growth rate during zebrafish fin regeneration. Development
132, 5173-5183.
[16] Lee, T., Hami, D., de Val, S., Kagermeier-Shenk, B., Wills, A.A., Black, B.L., Weidinger, G., Poss, K.D. (2009). Maintenance of blastemal proliferation by functionally
diverse epidermis in regenerating zebrafish fins. Developmental Biology 331, 270-280.
[17] Lopez-Olmeda, J.F., Sanchez-Vazquez, F.J. (2011). Thermal biology of zebrafish. Journal of Thermal Biology 36(2), 91-104.
[18] Manuel, M.B., Carmen, M. (2010). Dermoskeleton morphogenesis in zebrafish fins. Developmental Dynamics 239, 2779-2794.
[19] Nechiporuk, A., Keating, M.T. (2002). A proliferation gradient between proximal and
msxb-expressing distal blastema directs zebrafish fin regeneration. Development 129, 26072617.
[20] Pfefferli, C., Jazwinska, A. (2015). The art of fin regeneration in zebrafish. Regeneration
2(2), 72-83.
[21] Poss, K.D., Shen, J., Nechchiporuk, A., McMahon, G., Thisse, B., Thisse, C., Keating, M.T. (2000). Roles of Fgf signaling during zebrafish fin regeneration. Developmental
Biology 222, 347-358
Page 21 of 22
California University of Pennsylvania
Eric Moeller
[22] Poss, K.D, Nechiporuk, A., Hillam, A.M., Johnson, S.L., Keating, M.T.(2002). Mps1
definesa proximal blastemal proliferative compartment essential for zebrafish fin regeneration. Development 129, 5141-5149.
[23] Quint, E., Smith, A., Avaron, F., Laforest, L., Miles, J., Gaffeild, W., Akimenko, M.A.
(2002). Bone patterning is altered in the regenerating zebrafish caudal fin after ectopic
expression of sonic hedgehog and bmp2b or exposure to cyclopamine. Proceedings of the
National Academy of science of the United States of America 99, 8713-8718.
[24] Selever, J., Liu, W., Lu, M.F., Behringer, R.R., Martin, J.F. (2004). Bmp4 in limb
bud mesoderm regulates digit pattern by controlling AER development. Developmental
Biology 276, 268-279.
[25] Smith, A., Avaron, F., Guay, D., Padhi, B.K., Akimenko, M.A. (2006). Inhibition of
BMP signaling during zebrafish fin regeneration disrupts fin growth and scleroblast differentiation and function. Developmental Biology 229, 438-454.
[26] Sorensen, J.G., Kristensen, T.N., Loeschcke, V. (2003). The evolutionary and ecological
role of heat shock proteins. Ecology Letters 6, 1025-1037.
[27] Stewart, S., Gomez, A.W., Armstrong, B.E., Henner, A., Stankunas, K. (2014). Sequential and opposing activities of Wnt and BMP coordinate zebrafish bone regeneration. Cell
Reports 6(3), 482-498.
[28] Wehner, D., Cizelsky, W., Vasudevaro, M.D., Ozhan, G., Haase, C., Kagermeier-Shenk,
B., Röder, A., Dorsky, R.I., Moro, E., Argenton, F., Kühl, M., Weidinger, G. (2014).
Wnt/β-catenin signaling defines organizing centers that orchestrate growth and differentiation of the regenerating zebrafish caudal fin. Cell Reports 13(6), 467-481.
Page 22 of 22
Zebrafish”
An Honors Thesis
by
Eric Moeller
California, Pennsylvania
2019
Contents
1 Introduction
1
1.1
An overview of the fin regeneration process . . . . . . . . . . . . . . . . . . .
2
1.2
Molecular mechanisms underlying regeneration . . . . . . . . . . . . . . . . .
3
1.3
The effects of temperature on regeneration . . . . . . . . . . . . . . . . . . .
5
1.4
Heat-shock proteins and stress responses . . . . . . . . . . . . . . . . . . . .
6
2 Methods
7
2.1
Zebrafish care and maintenance . . . . . . . . . . . . . . . . . . . . . . . . .
7
2.2
Anaesthesia and amputation of the caudal fin
. . . . . . . . . . . . . . . . .
7
2.3
Tissue staining for analysis of fin branching . . . . . . . . . . . . . . . . . .
7
2.4
Immunohistochemistry to analyze differences in signaling proteins expressed
2.5
in regenerating fins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8
RNA analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8
3 Results
8
3.1
Ray branching . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8
3.2
Immunohistochemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
12
3.3
RNA analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
14
4 Discussion
15
California University of Pennsylvania
1
Eric Moeller
Introduction
The ability to regrow amputated limbs holds much interest in the field of biology. The
process of regeneration reactivates pathways that are used during initial limb development
as an embryo, which are conserved in humans. Research into regeneration may discover new
treatments that enhance human wound healing, possibly even allowing for the regrowth of
limbs. Regeneration follows a unique and complex process that is not often observed outside
of teleost fish and urodele amphibians. Zebrafish, for example, have a remarkable ability to
regenerate acutely damaged fins (Johnson and Weston, 1995), optic nerves (Goldman et al,
2001), and parts of their heart (Poss et al, 2000). This ability was first observed in 1786 by
the French scientist Broussonet who found that when zebrafish lost part of their caudal fin,
they were able to regrow them within 2-4 weeks (Pfefferli and Jazwinska, 2015).
In mammals, fibrosis is the dominant reaction to substantial injuries. This results in
the formation of scar tissue, which can cause discomfort and loss of function. In zebrafish,
however, this initial fibrin deposition is broken down to allow for regeneration. If regeneration
is inhibited, fibrin is not broken down and the healing ceases at scar formation. It has been
proposed that scarring precedes regeneration, and the proliferation of myocytes determines
whether wound healing will cease at scar formation or lead to regeneration (Poss et al, 2002).
Research into zebrafish regeneration allows scientists to more fully understand the mechanistic pathways involved in tissue regeneration. This understanding could have impacts
in many scientific fields through expanding our knowledge of genetics and cell signalling
(Gemberling et al, 2013). Novel discoveries could also aid in the development of medical
treatments if regeneration becomes understood fully enough to induce wound healing to
follow similar pathways in humans.
Page 1 of 22
California University of Pennsylvania
1.1
Eric Moeller
An overview of the fin regeneration process
The fin of teleost fish is mainly comprised of bony rays that support the structure of the
fin, inter-ray mesenchymal cells, and epidermal cells. Blood vessels and nerve axons extend
through both the rays and the mesenchymal tissue. The bony rays are maintained and created by osteoblasts, which function to deposit bone along the rays. The caudal fin fold is
stabilized by 16-18 bony rays that occassionally branch named lepidotrichia. The distal ends
of the rays, called actinotrichia, remain uncalcified while the rest of the structure is mineralized (Pfefferli and Jazwinska, 2015). In zebrafish, characteristic stripes of chromatophores
extend the length of the body, resulting in a dark striped pattern.
When the fin is amputated, regeneration begins quickly. Within a few hours, cells cover
the wound to protect it, forming a structure called the apical ectodermal cap which plays a
similar role to the apical ectodermal ridge in limb development. Within 1-2 days of wound
cap formation, dedifferentiated cells migrate underneath the epidermal wound cap and form
a white opaque mass called a blastema (Gemberling et al, 2012). For example, mature
osteoblasts have been found to dedifferentiate and migrate to form part of the blastema.
These cells are lineage-restricted, which has also been observed in amphibians. (Pfefferli
and Jazwinska, 2015; Quint et al, 2002). Blastema formation distinguishes regeneration
from limb development, however the rest of the outgrowth of the fin follows developmental
processes (Manuel and Carmen, 2010).
The blastema is formed from at least two lineages, osteoblasts and fibroblast-like cells
(Knopf et al, 2011). Cells within the blastema form two distinct groups known as a distal
blastema and a proximal blastema. The distal blastema proliferates slowly, providing a direction for the outgrowth and contributing daughter cells to the proximal blastema. The
proximal blastema proliferates rapidly and cells within it redifferentiate to produce the structures that were lost (Iovine, 2007).The cells of the blastema rapidly begin to migrate distally
from the site of amputation, and maintain a milky white appearance for the first few days
of tissue regeneration. This process continues, proceeding distally until the fin is reformed.
Page 2 of 22
California University of Pennsylvania
Eric Moeller
The regeneration process takes around 2-4 weeks depending on water conditions, and is not
affected by repeated amputations (Azevedo et al. 2011).
Figure 1: Diagram of the fin regeneration process (Pfefferli and Jazwinska, 2015)
1.2
Molecular mechanisms underlying regeneration
It has been proposed that the distal blastema acts as a signaling center during the regeneration process through the Wnt signaling pathway, which regulates epidermal patterning,
cell proliferation, and osteoblast differentiation through fibroblast growth factor (FGF) and
bone morphogenic protein (BMP) pathways (Wehner et al, 2014). Redifferentiation of the
osteoblasts in the proximal blastema requires a downregulation of the Wnt pathway by BMP
signaling, so the interplay between these two signaling pathways determines the dedifferentiation and redifferentiation of osteoblasts (Stewart et al, 2014).In development, FGF genes
function in wound healing, mesoderm induction, mature tissues/systems angiogenesis, and
the proliferation of fibroblasts, which fills up the wound space/cavity early in the wound healing process. FGF is a small secreted molecule that activates downstream signaling events in
nearby cells by binding to it’s cognate receptor, the FGF receptor. There are several FGF
genes and FGF receptors. FGF is expressed in mesenchymal cells underlying the wound
epidermis during blastema formation and in distal blastemal tissue during regenerative outgrowth (Poss et al. 2000).
Cells exppressing Fgfr1 create a signaling center that is required for the creation of the
proximal and distal blatema (Poss et al, 2002; Poss et al, 2000; Nechiporuk and Keating,
Page 3 of 22
California University of Pennsylvania
Eric Moeller
2002). FGF signaling in the distal blastema regulates cell proliferation in the proximal
blastema, while in the lateral basal epidermal layer it is required for bone differentiation and
is represented by the expression of Shh (Iovine, 2007). Shh signaling controls the patterning
of the regenerating tissue in a concentration-dependent manner. Shh active cells also secretes
Gre, which suppresses BMP signaling. BMP in turn inhibits FGF signalling, causing the
cells to stop growing (Selever et al, 2004). Shh and bmp2b can stimulate the secretion of
bone matrix within the blastema, possibly by influencing the differentiation of osteoblasts.
When Shh and FGF are close in proximity to each other, Shh inhibits the downregulating
BMP signal via Gre, allowing FGF to be expressed and growth to occur. As the tissues
move further apart, the Gre signal becomes less concentrated, and at a certain concentration
threshold the BMP signal becomes strong enough to inhibit FGF, causing the tissue to stop
growing (Freeman, 2000).
Figure 2: Diagram of pathways involved in limb formation (Freeman, 2000)
During fin regeneraion, Shh has been shown to be upregulated in the lateral basal epidermal layer around the area where the fin rays are regenerating. Other genes controlled
by Shh are induced in the basal epidermal layer and in nearby mesenchymal cells (Smith et
al, 2006). Expresison of shh or bmp2 between fin rays leads to upregulation of osteoblast
transcription factors which results in the formation of bone. This suggests that either Shh
Page 4 of 22
California University of Pennsylvania
Eric Moeller
or BMP is sufficient for bone formation (Quint et al, 2002). Knockout of Shh alone does
not result in a lack of bone formation, however chordin inhibition of BMP’s prevents the
expression of genes involved in the differentiation of osteoblasts, the development of mature
osteoblasts, and mineralization of the fin rays, which suggests that BMP is required for ray
development (Smith et al, 2006).
Both Sonic hedgehog and Indian hedgehog are activated in the blastemal tissue and
regulate blastemal proliferation, maintenance, and tissue growth (Iovine 2007). Interactions
with other signaling pathways including FGF and Wnt signaling has been shown to amplify
the regenerative response during fin regeneration (Lee et al. 2005). FGF activates and
regulates blastemal proliferation, causing extension of the tissue. It also controls epidermal
gene expression and maintains the expression of WNT signaling (wntb5), proximal Shh,
and lef1. It has been suggested that FGF may stimulate blastemal outgrowth through the
maintenance of Shh signaling, which is similar to their roles in limb development (Pfefferli,
2015).
1.3
The effects of temperature on regeneration
Zebrafish live in areas with temperatures that fluctuate often, and as such they can tolerate
a range of temperatures without lasting harm to the fish. In their natural habitat of north
east India, Bangladesh and Nepal, the lowest water temperature recorded was 14.2 ◦ C and
the highest was 33 ◦ C (Lopez-Olmeda and Sanchez-Vazquez, 2011). temperature stress is
only one form of stress which may have negative impacts on wound healing. In general,
stress is defined as a condition that disturbs the normal function of the biological system
or a condition that decreases fitness (Hoffmann and Parsons, 1991; Bijlsma and Loeschcke,
1997). If temperature stress affects wound healing, other sources of stress may also have
negative effects on wound healing in humans.
Page 5 of 22
California University of Pennsylvania
1.4
Eric Moeller
Heat-shock proteins and stress responses
Heat shock proteins are a class of chaperonin proteins that help proteins within a cell to fold
correctly. As the polypeptide chains are being transcribed, chaperonin proteins guide the
folding process. Apart from this function, molecular chaperones are involved in transport,
folding, unfolding, assembly, and disassembly of multi-structured units and degredation of
misfolded or aggregated proteins (Sorensen, Kristensen, and Loeschcke 2003). These proteins
also play a role in coping with oxidative and temperature stress, both of which cause proteins
to partially denature. If proteins refold incorrectly, they can become nonfunctional and
potentially harmful to the cell. To prevent this from occurring, chaperonin proteins also
work to guide the refolding process (Kim et al. 2013). When tissue is regenerating, protein
production is increased due to the need for cells to grow and proliferate quickly, resulting
in an increased demand for chaperonin proteins. Temperaure stress would also cause an
increase in demand for caperonin proteins, and it has been previously shown that when
there is a lack of chaperones, errors occur (Chaudhuri and Paul, 2006).
We hypothesize that fins regenerating in higher temperatureswould show errors in regeneration, and that the errors could be caused by an increased demand for heat-shock proteins
during temperature stress. Under temperature stress the chaperonin proteins are dealing
with the stress of the warmer temperatures, so more errors in protein folding could occur
within the rapidly dividing cells within the blastema. If developmental proteins involved in
cell to cell signalling are functioning abnormally, less signal would be received by cells and
they would not develop the overall tissue as precisely. Errors in cell signalling could cause
less organization in the overall tissue and lower protein expression of developmental signaling proteins within the tissue. If temperature stress increases the workload of chaperonin
proteins, there should be a significant upregulation of chaperonin gene expression in both
cold and warmer temperatures when compared to normal temperature subjects.
Objectives
• To determine if temperature stress affects regenerated fin morphology by examining
Page 6 of 22
California University of Pennsylvania
Eric Moeller
differences in fin branching under different temperature conditions
• To determine whether there are differences in the levels of key signaling signals used
during fin regeneration under normal conditions and under temperature stress
2
2.1
Methods
Zebrafish care and maintenance
Three groups of 5-15 fish were kept in 10 gallon tanks, each with different water temperatures
(22◦ C, 28◦ C, 34◦ C). The three tanks were maintained under the same light/dark conditions
and they had the same feeding regimine (1-2 times per day).
2.2
Anaesthesia and amputation of the caudal fin
Fish were caught and submersed in a 0.16% solution of Tricane (ms-222) until muscle activity
ceased, then were transferred to a sterile petri dish. Approximately half of the fin was amputated using a sterile razor, after which the fish were transferred to a recovery beaker until
they began to swim normally. Once swimming appeared normal, the fish were transferred
back to the appropriate tanks.
2.3
Tissue staining for analysis of fin branching
Fin segments were collected at 0dpa, 7dpa, or 14dpa and rinsed in phosphate buffered saline
(PBS). The segments were then placed in 95% ethanol on ice for1hr before being transferred
to fresh 95% ethanol at room temperature overnight. Bone and Cartilage stain solution of
0.02% alcian blue and 0.005% alizarin red was added and left overnight at room temperature
with rocking. The fins were rinsed in a series of glycerol/0.25% KOH solutions and stored
in 50% glycerol/0.25% KOH for further clearing and storage. Fins were mounted in glycerol
and ray branching data was collected with a compound microscope.
Page 7 of 22
California University of Pennsylvania
2.4
Eric Moeller
Immunohistochemistry to analyze differences in signaling
proteins expressed in regenerating fins
Fins were collected after 4dpa and fixed in 4% paraformaldehyde at 4◦ C overnight. The
fins were rinsed five times with phosphate buffered saline with 1% tween20 (PBS-tween),
incubated in normal horse serum (30 µl), then the primary antibody dilution was added
and incubated at 4◦ C overnight. Fins were treated with antibodies for Gre (1:500), Shh
(1:500), HSP-70, FGF4 (1:500) or ZNS-5 (1:200). Fins from all three temperature treatments
were labeled with ZNS-5, hedgehog, Gremlin and FGF. Fins from the normal and warm
temperature treatments were labeled with HSP-70. The fins were rinsed four times PBStween, then a biotinylated second antibody dilution was added (anti-mouse 1:200). The
biotinylated antibody was detected using a vecta-stain ABC kit. Staining was imaged using
a compound microscope.
2.5
RNA analysis
RNA was isolated using TRIzol (Invitrogen) or RNeasy (Qiagen). Twelve fins were cut
from each temperature and samples from the same temperature were pooled to increase the
amount of RNA. Absorbtion was measured to measure RNA levels. A Superscript III firststrand synthesis kit (Invitrogen) was used to convert the RNA into complimentary DNA
(cDNA). PCR was performed to amplify FGF3 transcripts, and the results were analyzed
using agarose gel electrophoresis.
3
3.1
Results
Ray branching
Fin ray branching in regenerated fins was examined after 14 days (14 dpa), in fins taken
from fish regenerating in cold, normal and warm temperatures. A one-way ANOVA showed
Page 8 of 22
California University of Pennsylvania
Eric Moeller
that the degree of fin ray branching seen after regeneration was significantly different in
cold and warm temperatures, compared to normal temperatures (F2,614=668.33; P¡0.0001).
Post hoc comparisons using the Tukey HSD test indicated a lower degree of ray branching in
regenerated fins in cold temperature conditions (M = 0.477, SD = 0.501) compared to fish
regenerating in normal temperature conditions (M = 1.054, SD = 0.424) at 14dpa. There
were also significant differences in ray branching between regenerated fins in the cold and
warm temperature conditions (M = 1.928, SD = 0.424), as well as a difference in branching
between regenerated fins under normal and warm temperature condition (Cold vs. Normal
P ¡ 0.01, Cold vs. Warm P ¡ 0.01, Normal vs. Warm P ¡ 0.01).
We also compared how the amount of ray branching had changed over time, by comparing
ray branching between 0dpa and 14dpa fish for the cold, normal, and warm conditions,
and found that there are significant differences in each group. Fins regenerating in cold
temperature conditions had a lower degree of branching at 14dpa (M = 0.477, SD = 0.500)
compared to fins at 0dpa in cold conditions (M = 1.689, SD = 0.619) (t = 21.01; df =
335.01; P ¡ 0.0001) (Figure 3). Fins regenerating in the warm temperature conditions has a
higher degree of branching at 14dpa (M = 1.928, SD = 0.423) compared to fins at 0dpa in
the warm conditions (M = 1.736, SD = 0.543) (t = -3.61; df = 234.2; P ¡ 0.001) (Figure 4).
The fins regenerating in the normal temperature conditions had a lower degree of branching
at 14dpa (M = 1.054, SD = 0.323) compared to fins in the normal temperature conditions
at 0dpa (M = 1.75, SD = 0.534) (t = 13.41; df = 233.98; P ¡ 0.0001) (Figure 5).
Page 9 of 22
California University of Pennsylvania
Eric Moeller
Figure 3: The degree of branching observed after 0dpa and 14dpa post-regeneration in
fins regenerating in the cold (21◦ C) conditions.
Figure 4: The degree of branching observed at both 0dpa and 14dpa post-regeneration in
fins regenerating in normal (28◦ C) conditions.
Page 10 of 22
California University of Pennsylvania
Eric Moeller
Figure 5: The degree of branching observed at both 0dpa and 14dpa post-regeneration in
fins regenerating in warm (34◦ C) conditions.
Figure 6: The percent change in branching before and after regeneration in cold, normal,
and warm temperatures. A positive percentage indicates that the number of fins with that
degree of branching increased post-regeneration, while a negative percentage indicates that
the number of fins with that degree of branching decreased post-regeneration.
Page 11 of 22
California University of Pennsylvania
3.2
Eric Moeller
Immunohistochemistry
1. FGF expression
The fin regenerating in the cold temperature conditions (a) showed a very low expression of FGF (shown in purple) compared to the fin regenerating in the normal
conditions (b). The fin regenerating in the warm conditions (c) showed a greater
amount of FGF expression than the fin regenerating in cold conditions, but showed
less expression compared to the fin regenerating in the normal condition.
(a) Cold 22◦ C
(b) Normal 28◦ C
(c) Warm 34◦ C
Figure 7: FGF expression in zebrafish caudal fins at 4dpa post-regeneration
2. ZNS-5 osteoblast expression
More osteoblasts were observed during regeneration at elevated temperatures (b) compared to the normal temperaures (a) at 4dpa (ZNS-5 shown in purple). No osteoblasts
were seen in the cold 4dpa regenerated fins (not shown).
(b) Normal 28◦ C
(c) Warm 34◦ C
Page 12 of 22
California University of Pennsylvania
Eric Moeller
Figure 8: ZNS-5 expression in zebrafish caudal fins at 4dpa post-regeneration. The
arrow indicates a labeled osteoblast
3. HSP-70 expression
A higher degree of staining was seen in the fins that regenerated in the warm temperature (b) compared to the normal temperature (a).
(a) Normal 28◦ C
(b) Warm 34◦ C
Figure 9: HSP-70 expression in zebrafish caudal fins at 4dpa post-regeneration.
4. Gremlin expression
In the cold 4dpa regenerated fins, the gremlin expression was low and seemed mainly
concentrated at the distal edge of the blastema. The normal 4dpa regenerated fins had
a similar amount of expression, however the majority of the Gre expression appeared
to be concentrated at the site of amputation as well as at tip of the blastema. The
warm 4dpa regenerated fins had a higher expression at the site of amputation as well
as at the tips of the fin folds in the blastema.
Page 13 of 22
California University of Pennsylvania
(a) Cold 22◦ C
Eric Moeller
(b) Normal 28◦ C
(c) Warm 34◦ C
Figure 10: Gremlin expression in zebrafish caudal fins at 4dpa post-regeneration.
5. Sonic hedgehog expression
Hedgehog expression appeared to be concentrated at the site of amputation as well as
along the apical edge of the blastema in the cold and normal 4dpa fins. The warm
4dpa fins had a lower expression along the site of amputation, but appeared to have a
greater expression at the fin tips.
(a) Cold 22◦ C
(b) Normal 28◦ C
(c) Warm 34◦ C
Figure 11: Sonic hedgehog expression in zebrafish caudal fins at 4dpa
post-regeneration.
3.3
RNA analysis
No FGF3 was seen in the negative control or in RNA extracted from fins regenerating in cold
conditions. Strong bands were seen in RNA from both the normal and the warm conditions,
Page 14 of 22
California University of Pennsylvania
Eric Moeller
as seen in figure 8.
Figure 12: Agarose gel electrophoresis of FGF3 RNA transcripts extracted from fish in
cold (22◦ C), normal (28◦ C), and warm (34◦ C) conditions.
4
Discussion
In investigating whether heat stress affects fin morphology after regeneration, we found
that there are differences in branching, protein expression, and visual characteristics. After
Page 15 of 22
California University of Pennsylvania
Eric Moeller
regenerating in the warm conditions, there is a greater amount of ray branching compared to
fins that regenerate under normal temperature conditions. This suggests that fin regeneration
is affected by temperature stress. In the cold conditions, we saw a lower amount of ray
branching compared to fins that regenerated under normal temperature conditions. We
believe this indicates a slower regeneration rate at cold temperatures rather than abnormal
fin regeneration. This temperature dependent rate decrease has been observed before the fin
rays that regenerated in the normal conditions also had a reduction in branching compared
to initial branching, which might indicate that the fins have not fully regenerated at 14dpa.
The regeneration process is typically completed after approximately three weeks, so the
process may still be occurring at 14dpa (Pfefferli and Jazwinska, 2015).
Along with these differences in branching, other morphological abnormalities were seen.
The rays appeared to have a rougher appearance and appeared thicker in the cold and warm
conditions compared to the normal conditions. The bands of melanophores which give the
zebrafish their characteristic striped appearance was darker in the warm conditions compared
to those of fish kept in the cold conditions. This is consistent with previous findings, which
show that the number of melanophores decreased under cold temperature conditions of 17◦ C
compared to zebrafish in normal temperature conditions of 26.5◦ C (Kulkeaw et al., 2011).
Fish in the warm tank were also more energetic than the fish in the cold conditions, however
they appeared to succumb to the effects of the tricaine in a much shorter time frame. These
observations further suggest that temperature stress is having an effect on regeneration and
could be an interesting avenue to pursue in the future.
In addition to ray organization, there appeared to be differences in bone ray thickness
between fish regenerating in normal and warm temperatures. To investigate these differences,
osteoblasts were observed. Cell signaling networks direct osteoblast progenitors to rebuild the
fin rays, so an increase in osteoblasts may cause an increase in bone deposition (Armstrong et
al., 2017). There appeared to be a greater number of osteoblasts in the warm temperatures
compared to fins in the normal temperatures (Figure 9). This may explain why the rays
Page 16 of 22
California University of Pennsylvania
Eric Moeller
were thicker and less organized. If there are more osteoblasts producing bone than normal, a
greater amount of bone deposition would occur, causing a thickening of the rays. Osteoblasts
were not observed in the cold-regenerating fins at 4dpa, again likely due to the delay in
regeneration at this temperature.
Given that differences in ray branching were observed, we wanted to determine the molecular basis of these abnormalities. To examine this, we observed the expression of signaling
molecules within the regenerating fins at 4dpa, early in the regrowth process. We first looked
at the expression of FGF, a protein that is thought to be a key signaling factor within the
blastema. FGF is expressed in mesenchymal cells underlying the wound epidermis during
blastemal formation and in the distal blastemal tissue during the regeneration process (Poss
et al. 2000). Because FGF is secreted from the tip of the blastema in a gradient, we expected to see the greatest amount of FGF expression at the tip of the fin, with a gradual
lessening of expression further away from the blastema. This expression pattern can be seen
in the fin regenerating in normal temperature conditions (Figure 8b) but cannot be seen in
fins regenerating in cold conditions at 4dpa (Figure 8a). The fin regenerating in the cold
condition did not show an appreciable amount of FGF expression, and the physical shape of
the fin tip was flat whereas the fin tips in the normal and warm had distinct ridges. Again,
this may have been due to the slower rate of regeneration under cold temperature conditions.
This could be confirmed by looking at FGF expression later in the regeneration process.
The fin in the warm condition did show FGF expression, however the FGF was not
expressed in the same pattern as in the normal temperature (Figure 8c). The expression
appeared patchy, with some areas of high concentration at the tip of the blastema and
some areas with no FGF expression. This suggests that temperature stress affects levels
of signaling molecules during regeneration, supporting our hypothesis. The differences in
the expression pattern of FGF could explain the differences in morphology that we saw
in fins regenerating under temperature stress. FGF regulates cell proliferation and bone
differentiation, so differences in FGF signaling could cause morphological differences in the
Page 17 of 22
California University of Pennsylvania
Eric Moeller
regenerated fin (Iovine, 2007). To confirm that the difference in FGF levels are not the
result of reduced gene expression, but are rather specifically due to reduced protein levels,
indicative of issues with protein folding, we examined levels of FGF transcripts. We saw
similar levels of FGF3 RNA in both the normal and warm conditions, however no expression
was observed in the cold condition or in the negative control. The RNA bands for FGF
in the normal and warm conditions appeared to be the same strength, indicating that the
fins in both conditions are producing the same level of RNA to create FGF3 proteins. This
indicates that gene expression is not affected by temperature at the level of transcription and
supports the idea that functional protein levels are being affected by temperature stress. If
the protein expression of FGF is lower in fins regenerating in warm temperatures compared
to normal temperatures, then the difference was occurring on the protein level and is likely
due to the lack of availability of chaperone proteins (heat-shock proteins) that normally
help ensure proteins are folded correctly. Temperature stress has been shown to cause an
increased demand for chaperonin proteins, and errors occur when there is an insufficient
supply to deal with the stress (Chaudhuri and Paul, 2006). The lack of RNA and protein
expression for FGF in the cold condition is likely due to the slower regeneration time under
lower temperatures rather than temperature stress.
To confirm that the fish were under temperature stress, we labeled a heat shock protein
that is known to aid cellular processes under temperature stress (HSP-70). We saw a greater
expression of HSP-70 in the elevated temperature conditions compared to the normal conditions (Figure 9), which indicated that fish in the elevated temperature were experiencing
a greater amount of stress.
The expression of another signaling protein, gremlin, was investigated. In development
this molecule acts as a concentration dependent inhibitor of BMP, which in turn is a concentration dependent inhibitor of FGF. As the Shh and FGF signaling centers move further
apart, gremlin becomes less concentrated. When gremlin concentration drops to a certain
point, BMP is no longer inhibited and can suppress FGF, causing the tissue to stop growing
Page 18 of 22
California University of Pennsylvania
Eric Moeller
(Freeman, 2000). We found that the expression of gremlin was lower in both the cold and
warm conditions compared to normal (figure 9a, c). A lower expression of gremlin may allow
BMP to inhibit FGF, which could cause the differences in FGF expression that is seen in
the cold and warm conditions. Sonic hedgehog expression appeared to be concentrated at
the site of amputation and at the tip of the fins in the cold and normal fins, with a lower
expression in the center of the regenerating fins. In the warm 4dpa fin, Shh expression
was greatest along the tip of the blastema and appeared to be greater throughout the fin
compared to the fins regenerating in normal and cold temperatures. Shh signaling regulates
blastemal proliferation and tissue growth (Iovine, 2007), so a greater amount of Shh expression in the warm temperatures could lead to differences during the regeneration process and
may explain the morphological differences that were seen.
If the amount of heat stress proteins were insufficient to deal with the combined stress of
increased temperature as well as the large increase in cell division and growth, some proteins
will become nonfunctional. Our results appear to support this, as there was a lower level of
FGF protein expression in the warm conditions without a difference in the amount of RNA
transcripts for FGF3. These results, along with the other differences observed, indicate that
temperature stress does cause morphological differences in regeneration.
References
[1] Armstrong, B.E., Henner, A., Stewart, S., and Stankunas, K. (2017). Shh promotes
direct interactions between epidermal cells and osteoblast progenitors to shape regenerated
zebrafish bone. Development 144, 1165-1176
[2] Azevedo, A.S., Grotek, B., Jacinto, A., Weidinger, G., Saude, L. (2011). The Regenerative
Capacity of the Zebrafish Caudal Fin Is Not Affected by Repeated Amputations. PLoS
ONE 6(7), 1-8.
Page 19 of 22
California University of Pennsylvania
Eric Moeller
[3] Bjilsma, R. and Loeschcke, V. (1997). Environmental Stress, Adaptation and evolution.
Birkhauser Verlag, Basel.
[4] Chaudhuri, T.K., and Paul, S. (2006). Protein-misfolding diseases and chaperone-based
therapeutic approaches. The FEBS Journal 273(7). 1331-1349.
[5] Matthew, F. (2000). Feedback control of intercellular signalling in development. Nature
408, 313-319.
[6] Gemberling, M., Bailey, T.J., Hyde, D.R., Poss, K.D. (2013). The zebrafish as a model
for complex tissue regeneration. Trends in Genetics 29, 611-620.
[7] Goldman, D., Hankin, M., Li, Z., Dai, X., Ding, J. (2001). Transgenic zebrafish for
studying nervous system development and regeneration. Transgenic Research 10, 21-33.
[8] Hoffman, A.A., and Parsons, P.A. (1991). Evolutionary Genetics and Environmental
Stress. Oxford University Press 4, 248
[9] Iovine, M.K. (2007). Conserved mechanisms regulate outgrowth in zebrafish fins. Nature
Chemical Biology 3, 613-618.
[10] Johnson, S.L., Weston, J.A. (1995). Temperature-Sensitive Mutations That Cause StageSpecific Defects in Zebrafish Fin Regeneration. Genetics 141(4), 1583-1595.
[11] Kim, Y.E., Hipp, M.S., Hayer-Hartl, A.B.M., and Hartl, U.F. (2013). Molecular Chaperone Functions in Protein Folding and Proteostasis. Annual Review of Biochemistry 82,
323-355.
[12] Knopf, F., Hammond, C., Chekuru, A., Kurth, T., Hans, S., Weber, C.W., Mahatma,
G., Fisher, S., Brand, M., Schilte-Merker, S., Weidinger, G. (2011). Bone regenerates via
dedifferentiation of osteoblasts in the zebrafish fin. Developmental Cell 20, 713-724.
Page 20 of 22
California University of Pennsylvania
Eric Moeller
[13] Kulkeaw, D., Ishitani, T., Kanemaru, T., Ivanovski, O., Nakagawa, M., Mizuochi, C.,
Horio, Y., and Sugiyama, D. (2011). Cold exposure down-regulates zebrafish pigmentation.
Genes to Cells 16, 358-367.
[14] Laforest, L., Brown, C.W., Poleo, G., Géraudie, J., Tada, M., Ekker, M., Akimenko,
M.A. (1998). Involvement of the sonic hedgehog, patched 1 and bmp2 genes in patterning
of the zebrafish dermal fin rays. Development 125(21), 4175-4184.
[15] Lee, T., Grill, S., Sanchez, A., Murphy-Ryan, M., Poss, K.D. (2005). Fgf signaling
instructs position-dependent growth rate during zebrafish fin regeneration. Development
132, 5173-5183.
[16] Lee, T., Hami, D., de Val, S., Kagermeier-Shenk, B., Wills, A.A., Black, B.L., Weidinger, G., Poss, K.D. (2009). Maintenance of blastemal proliferation by functionally
diverse epidermis in regenerating zebrafish fins. Developmental Biology 331, 270-280.
[17] Lopez-Olmeda, J.F., Sanchez-Vazquez, F.J. (2011). Thermal biology of zebrafish. Journal of Thermal Biology 36(2), 91-104.
[18] Manuel, M.B., Carmen, M. (2010). Dermoskeleton morphogenesis in zebrafish fins. Developmental Dynamics 239, 2779-2794.
[19] Nechiporuk, A., Keating, M.T. (2002). A proliferation gradient between proximal and
msxb-expressing distal blastema directs zebrafish fin regeneration. Development 129, 26072617.
[20] Pfefferli, C., Jazwinska, A. (2015). The art of fin regeneration in zebrafish. Regeneration
2(2), 72-83.
[21] Poss, K.D., Shen, J., Nechchiporuk, A., McMahon, G., Thisse, B., Thisse, C., Keating, M.T. (2000). Roles of Fgf signaling during zebrafish fin regeneration. Developmental
Biology 222, 347-358
Page 21 of 22
California University of Pennsylvania
Eric Moeller
[22] Poss, K.D, Nechiporuk, A., Hillam, A.M., Johnson, S.L., Keating, M.T.(2002). Mps1
definesa proximal blastemal proliferative compartment essential for zebrafish fin regeneration. Development 129, 5141-5149.
[23] Quint, E., Smith, A., Avaron, F., Laforest, L., Miles, J., Gaffeild, W., Akimenko, M.A.
(2002). Bone patterning is altered in the regenerating zebrafish caudal fin after ectopic
expression of sonic hedgehog and bmp2b or exposure to cyclopamine. Proceedings of the
National Academy of science of the United States of America 99, 8713-8718.
[24] Selever, J., Liu, W., Lu, M.F., Behringer, R.R., Martin, J.F. (2004). Bmp4 in limb
bud mesoderm regulates digit pattern by controlling AER development. Developmental
Biology 276, 268-279.
[25] Smith, A., Avaron, F., Guay, D., Padhi, B.K., Akimenko, M.A. (2006). Inhibition of
BMP signaling during zebrafish fin regeneration disrupts fin growth and scleroblast differentiation and function. Developmental Biology 229, 438-454.
[26] Sorensen, J.G., Kristensen, T.N., Loeschcke, V. (2003). The evolutionary and ecological
role of heat shock proteins. Ecology Letters 6, 1025-1037.
[27] Stewart, S., Gomez, A.W., Armstrong, B.E., Henner, A., Stankunas, K. (2014). Sequential and opposing activities of Wnt and BMP coordinate zebrafish bone regeneration. Cell
Reports 6(3), 482-498.
[28] Wehner, D., Cizelsky, W., Vasudevaro, M.D., Ozhan, G., Haase, C., Kagermeier-Shenk,
B., Röder, A., Dorsky, R.I., Moro, E., Argenton, F., Kühl, M., Weidinger, G. (2014).
Wnt/β-catenin signaling defines organizing centers that orchestrate growth and differentiation of the regenerating zebrafish caudal fin. Cell Reports 13(6), 467-481.
Page 22 of 22