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Characterization of midkine-a function in zebrafish heart regeneration

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Characterization of midkine-a function in zebrafish heart regeneration

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Content Characterization of Midkine-a function in zebrafish heart regeneration
By
Tim Tuai
A Thesis Presented to the
FACULTY OF THE USC KECK SCHOOL OF MEDICINE
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the Requirements for the Degree
Master of Science
(Biochemistry and Molecular Medicine)
August 2022
ii

Table of Contents
List of Figures iv

Abstract v

Chapter 1: Introduction 1
1.1: Introduction to heart disease 1
1.2: Midkine-a and its significance 2
1.3: Contrasting data regarding role of mdka 3
1.4: Cryoinjury vs. Amputation 4
1.5: Allelic Specificity Mutation Phenotypes 5
1.6 miRNA regulation of genes 6

Chapter 2: Materials and Methods 8
2.1: Animal Procedures 8
2.2 Genotyping 9
2.3: Histology 9
2.4: miRNA data analysis 10

Chapter 3: Results 12
3.1: Allelic specific differences of mdka mutants 12
iii

3.2: Genotyping mdka mutant zebrafish 13
3.3: Regeneration phenotype of mdka mutant zebrafish 15
3.4: Quantification of AFOG 17
3.5: miRNA analysis of mdka
mi5001
mutant zebrafish 18

Chapter 4: Discussion 24
4.1: Allele specific discussion 24
4.2: Cryoinjury in midkine-a mutant vs. wild-type zebrafish 25
4.3: miRNA results discussion 27

Conclusion and future directions 28

Bibliography 30

iv

List of Figures
Figure 1. Amputation vs. Cryoinjury induced myocardial infarction models 5

Figure 2. Allele specific mutation differences between mdka
mi5001
and mdka
cn105
models. 13

Figure 3. Genotyping of mdka mutant zebrafish 14

Figure 4. AFOG staining of mdka
mi5001
mutant zebrafish 16

Figure 5. Scar area quantification of mdka mutant hearts 17

Figure 6. Heatmap of differentially expressed miRNA 19

Figure 7. Predicted targets for miRNA downregulated in mdka
mi5001
mutants 21

Figure 8. Predicted target gene frequency in miRNA upregulated in mdka mutants at 7dpa 22

Figure 9. GO enrichment of dre-miR-93 23

Figure 10. GO enrichment of dre-miR-740 23

v

Abstract
Midkine is a heparin-binding growth factor that is conserved throughout animals and promotes
growth through effects such as proliferation, migration, and differentiation. In zebrafish,
Midkine-a (Mdka) was shown to be involved in epimorphic regeneration in zebrafish such as
caudal fin and retinal neuron regeneration. While mdka is a gene found to be up-regulated in
regenerating zebrafish hearts upon amputation, controversial data have been reported about its
role in the heart regeneration process. I systematically studied the functions of mdka using
different mutant alleles and injury models. I performed AFOG staining of the collagen scars to
compare mutant versus control zebrafish hearts. Furthermore, I compared micro RNA (miRNAs)
profiles in mdka mutants and controls that might reflect more subtle phenotypical changes.
Finally, I discuss the potential roles of mdka in the heart regeneration process.
1

Chapter 1: Introduction
1.1: Introduction to heart disease
Cardiovascular disease is the number one cause of human mortality worldwide. After a
heart injury, the damaged area is replaced with a fibrotic scar as a quick patch to repair the tissue,
but new muscle is not regenerated in adult mammals (Talman & Ruskoaho, 2016). This scarring
compromises the infrastructure of the heart and leads to heart failure due to poor contractibility
and ultimately leads to death. Zebrafish have shown regenerative capabilities in multiple organs
(Beffagna, 2019; Kroehne et al., 2011; Pfefferli & Jaźwińska, 2015), including the adult heart
(González-Rosa et al., 2017), which is in contrast to mammalian models such as mice where
regeneration of the heart only occurs in the neonatal stage (Porrello et al., 2011). Because of their
regenerative capabilities, available forward and reverse genetic tools, and ease of imaging adult
and developing hearts, zebrafish has become a prime model to understand the molecular
mechanisms of heart regeneration. One of the important regenerative processes is
revascularization - the formation of new coronary vessels after a heart injury has occurred and it
is critical for the myocardium to regenerate. Blood vessels are able to regenerate from the
existing vasculature in a process known as angiogenesis (Marín-Juez et al., 2016). For
capillaries, this regeneration can occur in ischemic situations, where insufficient blood flow
occurs and leads to myocardial hypoxia, where oxygen is deprived. This hypoxic signal will
induce angiogenesis (Krock et al., 2011) and ultimately form new capillaries that are associated
with pdgfrb+ pericytes. However, coronary arteries cannot regenerate by themselves, so it is
important to understand mechanisms of formation of the coronary artery.
2

1.2: Midkine-a and its significance
Midkine is a heparin-binding growth factor that is highly conserved throughout animals
and promotes growth through effects such as cell proliferation, migration, and differentiation.
Midkine is special due to its property of having multiple receptors, such as protein-tyrosine
phosphatase ζ (PTPζ) (T. Muramatsu, 2002), low density lipoprotein receptor-related protein (S.
Chen et al., 2007), anaplastic lymphoma kinase (Stoica et al., 2002), integrins (H. Muramatsu et
al., 2004), neuroglycan C (Ichihara-Tanaka et al., 2006), and notch2 (Huang et al., 2008),
allowing for a wide variety and range of influence. Induction of PTPζ, one of the more
recognized receptors of midkine, is able to affect major cellular responses due to MAPK/PI3K
signaling being involved in the downstream signaling of the receptor (Qi et al., 2001). Human
research of MIDKINE showed the gene to play a pivotal role in cancers (Filippou et al., 2020)
and a potential target as tumor markers. However, the midkine gene along with its other family
member pleiotrophin have also been seen to be related in functions relating to development and
inflammation (T. Muramatsu, 2002). In animal models, increased expression of Midkine in mice
has shown to show pro-angiogenic effects following cardiac injury (Sumida et al., 2010),
lowered expression of Midkine was shown to have reduced liver regeneration capabilities in
mice (Ochiai et al., 2004), and Midkine deficient mice displayed delayed degeneration and
regeneration after skeletal muscle injury (Ikutomo et al., 2014). The zebrafish genome contains
two midkine genes: midkine-a (mdka) and midkine-b (mdkb). In regeneration research, mdka has
been demonstrated to be an important factor in processes such as epimorphic regeneration (Ang
et al., 2020). In the heart regeneration process of zebrafish, mdka is a growth factor shown to be
induced (Lien et al., 2006) after the heart is subject to injury. Prior scRNAseq data identified
mdka being an upregulated gene in pdgfrb+ cells during heart regeneration in addition to having
3

similar patterns in gene expression in epicardium derived cells as pdgfrb. However, previous
research has shown that zebrafish homozygous mdka mutants (mdka
mi5001
) were insufficient in
preventing neither heart regeneration nor the manifestation of fibrotic scars when compared to
the control wildtype zebrafish. Instead, the scar depositions in mdka
mi5001
mutants were found to
be non-significant when compared to that of the wild-type zebrafish. Because mdka has shown to
be induced in heart injury models and highly expressed in epicardium and epicardium-derived
cells, it is important to understand whether mdka is required for epicardial cell closure over the
wound and revascularization of regenerating hearts.
1.3: Contrasting data regarding role of mdka
Controversial data has recently been published regarding the role of mdka in the heart
regeneration process. Using mdka
cn105
mutants and a cryoinjury model, it has been reported that
mdka was necessary for heart regeneration due to its role in regulating the fibrosis process. In
their study, mdka
cn105
mutants showed lowered cell proliferation leading to the prevention of
heart regeneration noted by the collagen scar formation and preservation (Grivas et al., 2021). In
contrast, other findings show that when using the traditional amputation method of ventricular
excision to induce injury to the heart, mdka
mi5001
mutant zebrafish were insufficient in producing
a heart regeneration defect phenotype; showing near full regeneration of the tissue ~30 days post
amputation (Kapuria et al., 2022). We aim to determine the phenotypic differences in heart
regeneration between mdka mutant alleles, as we believe that different reported phenotypes can
be caused by differences in alleles or injury type.

4

1.4: Cryoinjury vs. Amputation
Traditionally, the heart regeneration process is studied in zebrafish by the apex
amputation of the ventricle. Using this method, the heart bleeds until the rapid formation of a
fibrin clot assimilates, preventing the zebrafish from draining more blood. This fibrin clot is
replaced by new muscle in the following weeks, with an almost perfect recovery of the
amputated ventricle 30 to 60 days post injury. However, studying the effects of the model is
limited due to being solely based on tissue removal, rather than tissue damage such as the
inclusion of dead cardiomyocytes attached to the tissue. As a result, other methods such as
cryoinjury are now being used to induce tissue death and subject the heart to injury in order to
better simulate the effects on the heart that undergo myocardial infarctions. Unlike amputation,
cryoinjury affects cell types within the epicardium, endocardium, and coronary vasculature by
inducing a more severe apoptotic response. The method not only provides necrotic tissue to
remove, but the heart also undergoes permanent cardiac remodeling following injury not seen in
apex amputation models such as the ventricle size increasing, thickening of the injured wall, and
the formation of a more rounded ventricular shape (González-Rosa et al., 2017).
5

Figure 1. Amputation vs. Cryoinjury induced myocardial infarction models
Comparison of the two of the methods for simulating myocardial infarction effects in zebrafish.
(A) Model of the traditional method of cardiac injury (amputation of the ventricle), removing
~20% of the whole tissue. (B) Model of cryoinjury-induced myocardial infarction, using a
chilled probe to damage the ventricle of the heart, leaving dead cardiomyocytes in the process.
1.5: Allelic Specificity Mutation Phenotypes
Allelic specificity refers to the phenomenon where different mutant targeted sites on the
same gene may produce different phenotypes. It has been shown that within the same gene,
different mutant alleles resulted in varying levels of epistasis within yeast (Xu et al., 2012). One
of possible reasons for allele specific mutations reside in the location of the intended target site,
6

as mutation of an important part of the gene such as the domain may produce distinct effects than
the mutation of an exon at the end of the coding sequence (CDS).
The Crispr/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-
associated protein 9) technology is frequently used for its use in gene editing, using a single
guide RNA (sgRNA) to target specific sequences and create double stranded DNA breaks,
ultimately activating DNA repair by non-homologous end joining (NHEJ) and consequently
inserting or deleting nucleotides in the process (indels). While these indels that form produce
premature stop codons are likely to be degraded by nonsense-mediated decay which might
trigger genetic compensation (El-Brolosy et al., 2019) those that are not are subject to exon-
skipping. This is a phenomenon sometimes seen within mutant genes generated from
Crispr/Cas9 (Mou et al., 2017), where mutant mRNA in KP1/2 cell lines were not fully degraded
due to the mutated exon being “skipped”, leading to in-frame functional mRNA being translated
into functional protein. This phenomenon has also been seen in zebrafish models (Prykhozhij et
al., 2017), where a mutation in the gene pycr1a disrupted exonic splicing enhancers that lead to
one of its exons being skipped, producing an aberrant or truncated protein that may function
abnormally.
1.6 miRNA regulation of genes
MicroRNAs (miRNA) are non-coding RNAs that are ~22 nucleotides in length and are
able to play a role in regulating a gene’s expression pattern either by transcriptional repression or
mRNA degradation (Bartel, 2004). They are made mostly by being transcribed from DNA
sequences to create primary-miRNA and then subsequently processed to precursor miRNA and
7

mature miRNA where they can perform their duty in the cell. In most cases, miRNA functions as
gene suppressors (Ha & Kim, 2014), binding to the 3’ UTR region of mRNA and interfering
with the translation process. However, some miRNA have been found to show the opposite
effect under specific conditions (Vasudevan, 2012). miRNAs are important due to their ability in
regulating processes such as cell proliferation, differentiation, and signaling (Bartel, 2004, 2009).
Noting more specific cases, miRNAs have been found to play very extensive roles in regulation
such as tumor growth/angiogenesis (Fang et al., 2011), apoptosis (Kouri et al., 2015), and even
inhibition of metastasis (Shan et al., 2015). In zebrafish, miRNA have shown to regulate various
developmental processes (Bhattacharya et al., 2017; P. Y. Chen et al., 2005; Wienholds et al.,
2005).
Exosomes are small vesicles (40-100 nm) within the extracellular vesicle family that are
released from cells into the extracellular area, and are generally transported through bodily
fluids. miRNA have recently been found to reside in these exosomes (Montecalvo et al., 2012),
which differ from intrinsic cellular miRNA due to their ability to affect other cells. These
miRNA are also known as exosomal miRNAs, and are a subtype of miRNA that when packed
into exosomes, are secreted between cells and can have functions such as the ability to influence
cell functions in targeted cells (Kosaka et al., 2010). It has also been reported that the miRNAs
packaged into exosomes is not random and the miRNA that were found in exosomes were not
seen in the parental line (Mittelbrunn et al., 2011; Valadi et al., 2007), which leads us to believe
that investigating the expression analysis of miRNAs may give us clues to the any subtle
phenotypic changes found within the mdka mutant.

8

Chapter 2: Materials and Methods
2.1: Animal Procedures
Mdka
mi5001
(~1 year old) mutant zebrafish lines were maintained at Children’s Hospital
Los Angeles (CHLA) and were compared to wild-type AB strains. These fish are under standard
care by CHLA Animal care facility IACUC oversight. Zebrafish were anesthetized using
Tricaine solution and held on a dampened slotted sponge with its ventral side facing upwards.
Iridectomy scissors were used to create incisions in order to perforate the chest to access the
heart. Light pressure on the abdomen allowed for the ventricle to be exposed, which we removed
15-20% of the ventricular apex using the scissors aforementioned. The skin of the zebrafish was
not stitched back together following the surgery. Wounds of the zebrafish bled for 15-45 seconds
before clot formation and were healed 1-2 days post-surgery. Following the surgery, zebrafish
were placed in water and breathing was stimulated by squirting water over their gills. Zebrafish
returned to swim after 3 minutes and from the surgery, 90% of zebrafish survived, with the
deaths occurring the day of the surgery. After 7 days post amputation (recovery period), fish
were sacrificed to collect their extracellular vesicles for analysis.
For cryoinjury-induced myocardial infarctions, Mdka
mi5001
(~1 year old) mutant zebrafish
lines were maintained at Children’s Hospital Los Angeles (CHLA) and were compared to wild-
type Ekkwill strains. These fish are under standard care by CHLA Animal care facility IACUC
oversight. Zebrafish were anesthetized using Tricaine solution and were placed ventral side up
on a dampened slotted sponge. Iridectomy scissors were used to create an incision on the chest to
access the heart. Light pressure was applied to the abdomen for easy access of probing the
ventricular region. As described in (Harrison et al., 2019), cryoinjury was performed using a
9

cooled 0.8mm diameter spherical probe. The probe was cooled in liquid nitrogen for ~10 seconds
before being applied to the ventricular wall of the zebrafish heart. Warm water was used to heat
the probe following cryoinjury to allow easier removal of the probe. Fish that underwent surgery
were placed back in system water to recover. 5-7 fish of each group (mutant and wildtype) were
injured in order to account for variability in wound response in the heart regeneration process.
After 55-60 days post cryoinjury, both groups of fish were sacrificed and their hearts were
collected for injury analysis.
2.2 Genotyping
Zebrafish were anesthetized in Tricaine solution and 30% of the caudal fin was cut using
a sterilized surgical blade. DNA of zebrafish fins was extracted by subjecting individual fins to
50μL of NaOH and put into thermo cycler for 95°C for 30 minutes and incubated at 4°C. 5μL of
Tris HCl was added to the DNA extracted and then amplified using PCR using forward and
reverse primers for mdka
mi5001
5’-GCGATTAAATGGGAAGTGAATCC-3’ mdka-forward and
5’-CCTCCAAATTCTTTCTTCCAGTTG-3’ mdka-reverse and a Tm of 3-5°C below the melting
temperature of the primers. The PCR product was run on a 2% agarose gel to visualize the bands.
2.3: Histology
Acid Fuchsin Orange G (AFOG) Staining was used to stain tissue sections to distinguish
muscle from collagen and fibrin. Hearts were collected and fixed in 4% paraformaldehyde
overnight at 4°C. Following this, the heart was dehydrated by incubation in water, increasing
10

concentrations of EtOH, and 100% Toluene at room temperature. Additionally, the tissues were
incubated in 1:1, Toluene and Paraffin overnight with the next day being incubated in paraffin at
60-62 °C and subsequently embedded in paraffin. After the paraffin solidifies, 7 µM thick
sections are cut and dried at 37 °C on glass slides.
The paraffin sections on the glass-slides were de-waxed and the sections re-hydrated
shortly after. The sections were then incubated in preheated Bouins fixative and then
subsequently be incubated in room temperature. The sections were then washed with and
incubated in tap water. Following, tissue sections were incubated in 1% phosphomolybdic acid
and then rinsed with distilled water. Next, the sections were incubated in AFOG staining solution
and then rinsed with and incubated in distilled water. The tissue sections were then dehydrated,
cleared with toluene, and mounted with Cytoseal for imaging.
2.4: miRNA data analysis
Exosomal miRNA were analyzed using Partek Flow software (Partek® Flow® Software,
V10.0, 2020) . miRNAs were filtered by first trimming the adapter sequences from both the 3’
and 5’ end. Next the trimmed reads were aligned using bowtie and quantified dre-mirbase as the
annotation model. With the microRNA counts, noise was filtered by excluding features where
maximum <= 3. Counts were normalized to perform gene-specific analysis to search for
differentially expressed miRNA. Finally miRNA were filtered using the following variables:
p<=0.05, FDR step up (7dpa mutant vs. 7dpa control)<=0.05, FDR step up<=1 (7dpa mutant vs.
uninjured control), FDR step up<=1 (7dpa control vs. uninjured control), and fold change <-2 or
11

>2. Remaining miRNAs were plotted in a hierarchical clustering heat map for ease of
visualization.

Differentially expressed miRNA from the pipeline analysis were subjected to be analyzed
using miRmap web (Vejnar et al., 2013), a tool used to predict gene targets based on factors
including ΔG open, Probability exact, Conservation PhyloP, and miRmap score (suppression
likelihood) . We filtered out genes with a miRmap score of >90 and took the most common
target genes between differentially expressed miRNA groups. Following, we subjected the
miRNA predicted gene list with a miRmap score cutoff of >90 to GO term ontology, uncovering
biological processes that these miRNA may be associated with. For the case of dre-mir-93, we
opted to assume its mature miRNA form, dre-miR-93 for this analysis.

12

Chapter 3: Results

3.1: Allelic specific differences of mdka mutants
One of the considerations we had regarding the regeneration phenotype differences
observed between both of the mdka mutant models was by possible allelic specific phenotypes.
In zebrafish, mdka is a gene located in chromosome 7 and contains 5 exons. For the mdka
mi5001

model, the Crispr/Cas9 system was used to create a 19 base pair (bp) deletion in exon 3 and form
a premature stop codon (Nagashima et al., 2020). The mdka
cn105
model uses a similar
methodology to create its mutant, utilizing the Crispr/Cas9 in order to remove 16 bp while
adding 4 a bp fragment to also generate a premature stop codon (Grivas et al., 2021). While the
mutation sites in both models are alike, the amino acids generated from the mutation are
somewhat different. The most notable difference is that the mdka
cn105
mutant contains a 20 amino
acid change over the wild type gene while the mdka
mi5001
mutant is more conservative with the
original sequence, with a change of only two amino acids before reaching its stop codon.

13

Figure 2. Allele specific mutation differences between mdka
mi5001
and mdka
cn105
models.
The mutations found on mdka
mi5001
and mdka
cn105
are both located on exon 3 of the mdka gene.
(A) Different alleles of the mdka mutants generated using the Crispr/Cas9 system that resulted in
a premature stop codon. (B) Possible amino acid changes caused by the indels found in Figure
1A. Red hyphens indicate deleted bases, while red letters indicate added bases. Shortened bars
indicate exons in non-coding regions.
3.2: Genotyping mdka mutant zebrafish
To confirm the absence of mdka, we utilized PCR genotyping to amplify and visualize
the bands of the mdka
mi5001
mutant. DNA extracted from caudal fins of zebrafish was amplified
using the following primers: Mdka mutant zebrafish were identified using the following primers:
5’-GCGATTAAATGGGAAGTGAATCC-3’ mdka-forward and 5’-
CCTCCAAATTCTTTCTTCCAGTTG-3’ mdka-reverse using a denaturing temperature of 95°C
, annealing temperature of 55°C for 40 cycles, and synthesis temperature of 72°C. PCR products
14

were allowed to incubate in the thermo cycler at 10°C after amplification. The PCR products
were then visualized on a 2% agarose gel and the mutants were distinguished based on their size
relative to the wild-type control.

Figure 3. Genotyping of mdka mutant zebrafish
Caudal fins of mdka
mi5001
mutant zebrafish were amputated and their DNA was extracted by
incubation of NaOH. Forward primers 5’-GCGATTAAATGGGAAGTGAATCC-3’ and reverse
primers 5'-CCTCCAAATTCTTTCTTCCAGTTG-3' were used to detect both the homozygous
mutant (198 bp) and wild type (217 bp) band. Heterozygous bands depicted both 198 bp and 217
bp bands.

15

3.3: Regeneration phenotype of mdka mutant zebrafish
In order to determine whether mdka plays a role in the heart regeneration process of
zebrafish, we performed cryoinjury on both mdka
mi5001
mutant and wild-type zebrafish hearts to
induce the cardiac regeneration process in zebrafish. After the regeneration period of 55-60 days
post injury (dpi), we sacrificed the zebrafish to collect their hearts and embed them in paraffin.
Following, we sectioned the hearts and after mounting, stained with AFOG solution of both heart
models to visualize the scar area post regeneration. AFOG stains the collagen dark blue, fibrin
red, and cardiac must with other tissue brown.

16

Figure 4. AFOG staining of mdka
mi5001
mutant zebrafish
Tissue sections of mdka mutant and wild-type zebrafish hearts 55-60 dpi. (A- A’) AFOG staining
of the wild-type zebrafish heart after cryoinjury induced myocardial infarction. Hearts were
allowed 55-60 days after their injury type in order to fully facilitate the regeneration process.
Collagen depositions are stained in blue while fibrin is depicted in red. (B- B’) AFOG staining of
cryoinjury models of mdka mutant zebrafish heart 55-60dpi.

17

3.4: Quantification of AFOG
Supplementing the data in Figure 10, we quantified the AFOG data to understand from a
numerical perspective the differences in scar area. We found that the relative scar area sizes (scar
area/ventricle area) between wild-type and mdka mutant models 55-60 dpi were significant
(p<0.005) using an unpaired two tailed t-test.

Figure 5. Scar area quantification of mdka mutant hearts
Comparison of the relative scar areas between the amputation and cryoinjury models of
mdka
mi5001
mutant zebrafish heart. Scar areas were measured freehand using Adobe Photoshop to
measure both the collagen deposition and the ventricle area in pixels. Statistical analysis shows
that the difference in scar area was found to be significant (p<0.005) using an unpaired two tailed
t-test, n=3 for each group.
18

3.5: miRNA analysis of mdka
mi5001
mutant zebrafish
We were also interested in any possible differentially expressed miRNA found in mdka
mutant zebrafish hearts, as miRNA are known to fine tune gene expression and produce subtle
phenotypic changes. To test this phenomenon, mdka
mi5001
zebrafish were amputated to induce
regeneration of the heart and their exosomal miRNA were sequenced 7 days post amputation
(dpa). Following this, miRNAseq analysis was performed using Partek Flow software in order to
determine differentially expressed miRNA between mdka mutant, wild type zebrafish, and
uninjured control. In order to ensure a significant enough difference between the miRNA, we
opted to filter out miRNA with non-significant values and high false discovery rates, as well as
the lower magnitude fold changes between the LSMean values. The resultant genes were then
plotted on a hierarchical clustering heat map, as shown in Figure 12.

19

Figure 6. Heatmap of differentially expressed miRNA
Relative expression of different miRNA secreted by the heart in 7dpa wild type, 7dpa mutant,
and uninjured hearts. Hearts were subjected to miRNAseq and then analyzed using Partek Flow
software. miRNA containing significance level p<0.005, FDR step up<0.05, and a fold change
magnitude of 2 were filtered to ensure significant results and prevent false positives.

With the miRNA list, we opted to look further into select miRNAs that seemed
interesting, most notably those that either showed increased expression in 7dpa WT when
compared to uninjured control and 7dpa mutant and the miRNA group that had increased
expression in 7dpa mutant when compared to both the WT control groups (7dpa and uninjured).
20

We used miRmap web (Vejnar et al., 2013) to search for likely target gene candidates that the
differentially expressed miRNA were affecting. We opted to use the miRmap scoring system in
order to filter our candidate genes, which is a parameter of likelihood for suppression. Using a
miRmap score of >90 to filter out genes, we accumulated the most common predicted target
genes from the differentially expressed miRNA to better understand what processes these
miRNA were affecting. In Figure 13, we show the most frequently predicted targets for
differentially expressed miRNA that were down-regulated in 7dpa mdka mutants when compared
to the 7dpa wild-type control group.

21

Figure 7. Predicted targets for miRNA downregulated in mdka
mi5001
mutants
Predicted gene target for differentially expressed miRNA showing down-regulation in mdka
mutants upon amputation when compared to 7dpa WT control. Common gene targets were
filtered between miRNA using a miRmap score cutoff of >90.

We were also interested in those miRNAs that showed up-regulation in mdka mutants
when compared to the 7dpa WT control group. The most frequent predicted target genes are
listed in Figure 14 below.

22

Figure 8. Predicted target gene frequency in miRNA upregulated in mdka mutants at 7dpa
Predicted gene target for differentially expressed miRNA showing up-regulation in mdka
mutants upon amputation when compared to 7dpa WT control. Common gene targets were
filtered between miRNA using a miRmap score cutoff of >90.
To understand what biological processes were being affected by the miRNAs found, we
also subjected the predicted gene targets in individual miRNA to gene ontology enrichment
analysis (Mi et al., 2019). We chose one miRNA from each group to display GO enrichment,
with dre-miR-93 representing those miRNA with increased expression in 7dpa WT and dre-miR-
740 representing miRNA with increased expression in 7dpa mdka mutant zebrafish. We opted to
use a miRmap score cutoff of >90 to get a broader view of the processes affected. GO terms
relating to the same processes tree were color coded in the figures shown below.

23

Figure 9. GO enrichment of dre-miR-93
GO term enrichment of zebrafish miRNA dre-miR-93. dre-miR-93 is a miRNA found to
be upregulated in 7dpa WT control EVs when compared to that of mdka mutant. GO-terms were
selected using the Fisher’s Exact test and Calculate FDR correction.

Figure 10. GO enrichment of dre-miR-740
GO term enrichment of dre-miR-740 in zebrafish hearts. dre-miR-740 is a miRNA found to be
upregulated in mdka mutant EVs when compared to 7dpa WT control. GO-terms were selected
using the Fisher’s Exact test and Calculate FDR correction.

24

Chapter 4: Discussion
4.1: Allele specific discussion
Initially, the project was expected to include another mdka mutant zebrafish using a
different targeted mutation site; however, due to time constraints and extrinsic issues it was not
feasible to replicate the same experiments under the same conditions. The two mdka mutant
alleles in this study, mdka
mi5001
and mdka
cn105
, use the Crispr/Cas9 system in order to generate a
premature stop codon by indel formation. Because both of the indels created in mdka
mi5001
and
mdka
cn105
zebrafish mutants reside very closely on exon 3 of the CDS, we believe that the
phenotypic difference is not due to exon skipping, as both resultant proteins would more than
likely produce the same effects. Both of the models share similar mutation sites, with some
differences being present in their resulting changed amino acid formation, albeit very little.
While even a single amino acid change can result in drastic consequential effects such as sickle
cell anemia (Fitzsimmons et al., 2016), the proposed amino acid changes in the mdka
cn105
model
would have the most likely means of forming an aberrant protein, as the addition of proline and
glycine amino acids would likely disturb the alpha-helix secondary structure (Jacob et al., 1999).
However, both the mdka
mi5001
and mdka
cn105
studies have verified the mRNA degradation to be
significant, so in the case of any aberrant proteins due to the proline disturbing alpha helix or
possible exon skipping, the likelihood and resultant effect would be minimal.

We must also mention the possibility of genetic compensation induced by nonsense
mediated decay. In zebrafish, it has been seen that mutants of certain genes such as vegfaa was
enough to trigger a compensatory response from the gene paralogue vegfab (El-Brolosy et al.,
25

2019). While mdk a’s paralogue midkine-b was not found to be upregulated in the absence of
mdka (Kapuria et al., 2022), other genes can still mediate the compensatory effect (Buglo et al.,
2020). Because mdka is unique in its ability to have multiple receptors and affects multiple
downstream targets such as the MAPK and PI3K pathway in its human homologue MIDKINE,
we cannot completely rule out the chance that there may still be a compensatory mechanism in
the absence of mdka. This compensatory effect due to our allele may be the reason why we see a
lowered phenotypic effect compared to that of other labs’ experiments, or may be pertaining to
the mdka gene as a whole and why we do not see the scarring occur in amputation models when
compared to cryoinjury. To sum, more research would be needed to conclude whether this is an
allele-specific effect. Some of these future experiments include things such as the addition of
another mdka allele and qPCR analysis of that allele’s mRNA presence alongside exonic reads to
ensure there is no exon skipping present.
4.2: Cryoinjury in midkine-a mutant vs. wild-type zebrafish
Successful regeneration of cardiomyocytes in zebrafish is typically denoted by the lack of
collagen and fibrin deposition remaining in the tissue post regeneration (Simões et al., 2020), as
zebrafish are able to remove the transient fibrin clot with new regenerated tissue (Major & Poss,
2007). It is seen in Figure 11 that although the collagen and fibrin deposits are relatively low
phenotypically compared to that of the whole tissue, the quantification shown in Figure 12 shows
that the depositions of collagen were consistent within mdka mutants, and control wild-type
zebrafish had little to no scar formation, indicating full regeneration of the tissue after cryoinjury.
In contrast to this, mdka mutants undergoing apex amputation were found to show non-
26

significant amounts of collagen deposition between the mutant and control models (Kapuria et
al., 2022). Due to the differences in phenotypes we see between the two models we believe that
the regeneration defect found within mdka mutants is likely to be cryoinjury-specific. We can
attribute this to some of the factors seen in cryoinjury models not found in amputation, such as
more severe cell death within the ventricular wall (Chablais et al., 2011), leaving of necrotic
tissue, and possible permanent changes such as cardiac remodeling (González-Rosa et al., 2017).
In order to better understand this phenomenon, future experiments would include scRNAseq
comparing cryoinjury and amputation models of mdka mutants to see why these regeneration
differences occur, whether it be from cellular composition differences or differential gene
expression of individual cell types. Addressing the relatively “low” collagen scarring found in
Figure 11, we can attribute this to the technical aspects of the cryoinjury, as some factors may
influence the severity of phenotype observed. Factors such as size of the zebrafish, probe
duration, and individual experience may all be variable between experiments. It is a possibility
that what we are seeing is not due to mdka interacting with cryoinjury-induced myocardial
infarction, but rather a phenotype due to excess cryoinjury probing. We would like to conclude
with this that while the scar sizes between wild-type and mdka mutant zebrafish were statistically
significant, phenotypically we do not believe it is significant, at least in comparison to other
models seen such as in (Grivas et al., 2021). Therefore more studies may be needed to make a
more firm conclusion regarding the role of mdka during regeneration, such as the testing of a
third mutant allele.

27

4.3: miRNA results discussion
We were also interested in the miRNA of the mdka mutants, as miRNA is known to fine
tune the regulation of gene expression resulting in more subtle phenotypes. Our miRNA analysis
revealed two groups of interest: miRNA that showed up-regulation in 7dpa WT compared to the
mutant and WT, as well as miRNA that were up-regulated within mdka mutants but down-
regulated in both WT uninjured and 7dpa WT zebrafish. Use of the miRmap software allowed us
to find potential target genes commonly found between the miRNA that were likely to be
affected.
In the miRNA group found to be upregulated in WT 7dpa zebrafish but not in mdka
mutants, the TRPS1 gene was a gene that stood out to us. TRPS1 has been shown to have a role
in the regulation of cell cycle progression and cell proliferation (Wu et al., 2014) and in diseases
such as breast cancer (Hu et al., 2018) . Knowing this, it could be speculated that when this gene
is silenced/down-regulated, as the miRNA were expressed more in 7dpa WT zebrafish, cell
proliferation would be able to flourish and regenerate the lost tissue. Conversely, in the uninjured
and 7 dpa mdka mutant models, we can attribute the lowered expression of miRNA to the
lowered cell proliferation in both cases. When we apply this logic to mdka mutants, one possible
scenario is that because they do not emit this miRNA to lower the expression of TRPS1 is the
reason why we see the sustained scarring of the heart after amputation, as the cell cycling
process would be suppressed, leading to lowered cell proliferation.
In the miRNA group that was raised in mdka mutants compared to that of WT 7dpa, we
found genes such as notch3 and mmp14b to be potential targets for these miRNAs. Notch
signaling is known to have a role in cardiomyocyte proliferation during zebrafish heart
28

regeneration (Zhao et al., 2014) . Alongside this, notch3 as a gene is necessary for angiogenesis
(Liu et al., 2010), and is essential for vascular smooth muscle cells formation (Volz et al., 2015;
Wang et al., 2008). Knowing these basic roles of notch3, seeing it in the common gene target list
of the miRNA is interesting because it correlates with what we are seeing in the potential
phenotype of mdka mutant zebrafish. We can speculate the effects again to say that the down
regulation of the gene from the miRNA would lead to limited revascularization of the heart after
amputation, as there would be lowered cell proliferation migrating to the injured area.
Conclusion and future directions
The focus of this study was to address some of the variables in reported phenotypes
shown in mdka mutants’ mdka
mi5001
and mdka
cn105
, as well as to discover possible other roles of
mdka. I determined that there was a statistically significant regeneration defect shown between
mdka
mi5001
mutants and the wild-type zebrafish when myocardial infarction is induced by
cryoinjury. In addition to the cryoinjury data, I also performed data analysis on the exosomal
miRNA of mdka
mi5001
mutant zebrafish. Using Partek Flow software, I determined differentially
expressed miRNA between 7dpa WT and mdka
mi5001
mutant zebrafish and with those miRNA,
found common predicted gene targets that were of interest such as TRPS1 and notch3.
I would like to address that while the quantification showed significance, phenotypically
the relative scar area was low, and the statistical significance primarily comes from the wild-type
zebrafish showing full/near full regeneration. For this reason is why I would propose for the
validation of the data through more experiments. Because we only used one allele to test against
the reported phenotype in the de la Pompa lab (Grivas et al., 2021), a third allele mdka mutant
29

should be tested with the same parameters as the mdka
mi5001
mutant. The benefit of this proposal
is that we were already in the process of creating homozygous mutants of this third allele, but
could not replicate the same experiments due to the time constraints of the study. This third mdka
mutant will allow us to make more confident conclusions regarding the differences in
phenotypes seen, rather than speculation of possibilities.
To fully understand the role of mdka in zebrafish, some other future experiments may be
performed to further the project. One of these experiments is to do scRNAseq of tissue from
cryoinjury and amputation models to give us further clues as to the mechanism on why these
regeneration differences occur, whether it is from cellular composition differences or differential
gene expression of individual cell types. Previous AFOG staining results showed signs of
thinning on both the outer epicardial layers and inner trabeculations of the heart (Kapuria et al.,
2022), indicating that this may be an intrinsic cardiomyocyte problem. Whole mount imaging
will allow us to visualize cardiovascular markers in mdka mutants leading to possible
regeneration defects in developing and adult zebrafish. We can also follow up on the miRNA
data by either screening the activity of predicted target genes or we can silence the miRNA itself
using shRNAs or the Crispr/Cas9 system and verify the lingering collagenous scarring in the
heart using both amputation and cryoinjury induced myocardial infarctions.

30

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Abstract (if available)

Abstract Midkine is a heparin-binding growth factor that is conserved throughout animals and promotes growth through effects such as proliferation, migration, and differentiation. In zebrafish, Midkine-a (Mdka) was shown to be involved in epimorphic regeneration in zebrafish such as caudal fin and retinal neuron regeneration. While mdka is a gene found to be up-regulated in regenerating zebrafish hearts upon amputation, controversial data have been reported about its role in the heart regeneration process. I systematically studied the functions of mdka using different mutant alleles and injury models. I performed AFOG staining of the collagen scars to compare mutant versus control zebrafish hearts. Furthermore, I compared micro RNA (miRNAs) profiles in mdka mutants and controls that might reflect more subtle phenotypical changes. Finally, I discuss the potential roles of mdka in the heart regeneration process.

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Expression pattern analysis of coronary and lymphatic vessel genes in zebrafish and mice

Asset Metadata

Creator Tuai, Tim (author)

Core Title Characterization of midkine-a function in zebrafish heart regeneration

School Keck School of Medicine

Degree Master of Science

Degree Program Biochemistry and Molecular Medicine

Degree Conferral Date 2022-08

Publication Date 07/18/2022

Defense Date 05/23/2022

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag Amputation,analysis,cardiomyocyte,cryoinjury,Heart,mdka,midkine,midkine-a,miRNA,OAI-PMH Harvest,Regeneration,repair,zebrafish

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Subramanyan, Ram Kumar (committee chair), Lien, Ellen (committee member), Xu, Jian (committee member)

Creator Email tuai@usc.edu,wasabimaro@csu.fullerton.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-oUC111372149

Unique identifier UC111372149

Legacy Identifier etd-TuaiTim-10840

Document Type Thesis

Format application/pdf (imt)

Rights Tuai, Tim

Type texts

Source 20220718-usctheses-batch-954 (batch), University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the author, as the original true and official version of the work, but does not grant the reader permission to use the work if the desired use is covered by copyright. It is the author, as rights holder, who must provide use permission if such use is covered by copyright. The original signature page accompanying the original submission of the work to the USC Libraries is retained by the USC Libraries and a copy of it may be obtained by authorized requesters contacting the repository e-mail address given.

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA

Repository Email cisadmin@lib.usc.edu