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Exploring the role of vegfr transcripts during vascular development in the zebrafish embryo
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Exploring the role of vegfr transcripts during vascular development in the zebrafish embryo
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Copyright 2021 Maria Eleni Dimotsantou
EXPLORING THE ROLE OF vegfr TRANSCRIPTS DURING
VASCULAR DEVELOPMENT IN THE ZEBRAFISH EMBRYO
by
Maria Eleni Dimotsantou
A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(Biomedical Engineering)
May 2021
ii
ACKNOWLEGEMENTS
First and foremost, I would like to express my deep and sincere gratitude to my PhD
research advisor and committee Chair, Dr. Scott E. Fraser Ph.D, (Provost Professor of Biological
Sciences and Biomedical Engineering) and my science mentor / committee member Dr. Le A.
Trinh, Ph.D (Associate Research Professor of Biological Sciences), for their endless support
during this academic journey. Without their invaluable guidance and scientific mentoring, this
academic journey would have never been as educational and productive. But most of all, I am
grateful to be part of this academic family, despite the continuous PhD research challenges over
the years. A family that welcomed me in 2012 at Caltech, we transitioned together to USC and I
continued as part of them up to the moment of my graduation. I am proud to be part of the Fraser
Group since the very first moment of its creation at USC in 2013.
Secondly, I would like to thank my PhD Thesis Committee. Dr. Carolyn M. Phillips, Ph.D
(Assistant professor of Biological Sciences) for inspiring me to explore and better understand
mRNA processing bodies by diving deeper into the current biological questions. Dr. Megan L.
McCain Ph.D, (Chonette Early Career Chair and Assistant Professor of Biomedical Engineering)
for the inspiring conversations, stimulating questions and mentoring during my PhD and Dr.
Stacey D. Finley, Ph.D (Gordon S. Marshall Early Career Chair and Associate Professor of
Biomedical Engineering and Biological Sciences) for mentoring and unique engineering questions
combining translational biology and chemical engineering approaches. I am extremely proud to
have a PhD committee consisted by these exceptional women, which serve as scientific role
models to all current and upcoming female graduate students in Science and STEM. Thank you
very much, you are a continuous inspiration, and I would be forever grateful that I met you.
iii
Thirdly, I would like to express my sincere gratitude to all my USC colleagues and friends
who contributed directly or indirectly to this PhD research. Dr. Francesco Cutrale. PhD (Associate
Research Professor of Biomedical Engineering) for his insightful comments and allowing me to
use his newly published HYSP software, Dr. Chili Chiu (Imaris, Bitplane) for all the technical
support, Daniel Koo (BME graduate student) for helping me troubleshoot Matlab codes extracting
statistical data from Imaris interface and Valerie Thomas (MCB graduate student) for all countless
scientific discussions, emotional support and PhD presentation feedback. I would like to offer my
special thanks to Dr. Simon Restrepo & Dr Sara Restrepo-Vassalli for their wonderful
conversation and support at the Fraser lab, Dr. William Dempsey for being a unique scientific
model and dear friend since my first years at Caltech and Dr. Carol Readhead for being an
academic light during difficult times. I am sincerely grateful for your love and support during these
PhD years.
Furthermore, special thanks to BME colleagues. Dr. Andrey Andreev and Dr. Sara Madaan
for scientific conversations and for being there since the first day of our graduate school life at
USC. Dr. Julio Villalon Reina and Ivan Alberto Trujillo, for being the best scientific partners in
crime that anyone could ask during the BME years. Your name will always be affiliated to the
strongest and most pleasant memories during USC graduate school years. Extending my gratitude
to all friends and colleagues from Fraser group, all BME and USC family. Special thanks to
William Yang for all this support as BME research administrator during the final stages of the PhD
who has been the catalyst for this final PhD year. Never forgetting the support as well from
Mischalgrace Diasanta who was previously holding the same BME position.
On the other hand, I am never forgetting the inspiration coming from my Caltech
colleagues, Dr. Ryan Henning and Dr. Joe Varghese, Dr. Vanessa Eguavoen, my best friends, and
iv
biggest emotional supporters at Caltech as well as Dr. Mark Harfouche . Extra thanks to Dr. Ann
Cheung for bring an exceptional scientific mentor, and Dr. George Tolomiczenko for all his
support and scientific discussions. Special thanks to Dr. Jelena Culic Viskota for being an
academic “sestra” since the very first day of graduate school orientation at Caltech. Special
academic advisors who supported this academic journey from undergraduate and graduate years.
Dr. Constantinos Vayenas (UPatras), Dr. Spiridon Pandis (UPatras, Carnegie Mellon), Dr.
Dimitrios Spartinos (UPatras), Dr. Amnon Yariv (Caltech), Dr. Harry Gray (Caltech), Dr. Rob
Phillips (Caltech) and Dr. Julie Kornfield (Caltech).
Last by not least, this PhD Thesis would have never been concluded without the
unconditional love and support of my undergrad friends Dr. Elina Karnezi, Dr. Antonis Tasoglou,
Dr. Constantinos Anagnostopoulos and Akis Angelis throughout the years. Special thanks for all
the emotional support and empowerment of my friends Miselina Lemisiou, John Samaras,
Katerina Kalliadi, Marios Theodorou, Marianna Varviani, Sofia Alexiou, John Ioannidis,
Anastasios Papapostolou, Elena Liakou, Filippos Tsapekis and Joanna Kalafatis. Special thanks to
Alice Dimotsantos and to my beloved Angelo Dimotsantos, who is no longer with us.
Finally, all my personal dedication and inspiration during this journey was coming from
my family; father Spiro Dimotsantos, mother Maro Dimotsantou and brother George Dimotsantos.
I was blessed to have all their endless love and support.
v
TABLE OF CONTENTS
Acknowledgements.……………………………………………………………………………....ii
List of Tables...………………………………………………………………………………….vii
List of Figures.………………………………………………………………………………….viii
Abbreviations...…………………………………………………………………………………....x
Units.………....………………………………………………………………………………….xii
Abstract……....………………………………………………………………………………….xiii
Chapter 1: General Background ………………………………………………………...................1
1.1 Vasculogenesis, angiogenesis: Two important morphological processes.............1
1.2 Vascular morphogenesis and angiogenesis in zebrafish.......................................6
1.3 Critical regulators during vascular development.................................................12
1.4 VEGFR2 receptor endocytosis during angiogenesis............................................17
1.5 Known association patterns of VEGFRs with Notch signaling and regulation of
vessel morphogenesis in zebrafish............................................................................22
1.6 Introduction to in situ Hybridization Chain Reaction (HCR)..............................30
Chapter 2: Methods and Techniques……………………………………………………….…......36
2.1 In situ Hybridization Chain Reaction Methods and Protocol..............................36
2.2 Confocal Microscopy and Imaging Conditions...................................................53
2.3 Background on Hybrid Unmixing (HYSP)..........................................................57
2.4 Data Analysis Pipeline and Methods of Image Rendering (Imaris)....................62
2.5 Mathematical Background of Graphical Representations....................................73
Chapter 3: Exploring the role of vegfr1, vegfr2 and vegfr3 transcripts during angiogenesis and
vasculogenesis ……………………………………………………….…....................77
3.1 Introduction.........................................................................................................77
3.2 Multiplex HCR labelling of vegfr transcripts in the developing vasculature........80
3.3 Subcellular transcript localization in WT and dnm1l mutant background............89
3.4 Following the Spatiotemporal of vegfr1, vegfr2 and vegfr3 transcripts during 1
st
and 2
nd
wave of angiogenesis.....................................................................................97
3.5 Nuclei Dots: Tails of active transcription sites in the nucleus of cells in all vessel
types........................................................................................................................109
3.6 Conclusions.......................................................................................................119
Chapter 4: Future Directions………………………………………………………………........123
4.1 Importance of studying vegfr transcripts and its receptor dynamics in model
organisms................................................................................................................123
4.2 Membraneless organelles, P-bodies and potential association with vegfr transcript
regulation................................................................................................................131
vi
4.3 Current methods that allow selective mutation on important parts of vegfr
transcripts for expanding current PhD Thesis..........................................................134
4.4 Translational approaches: From studying model organisms to new anti-
angiogenic drug development.................................................................................135
Bibliography....…………………………………………………………………………………136
vii
LIST OF TABLES
Chapter 1: General Background…………………………………………………………….….N/A
Chapter 2: Methods and Techniques……………………………………………………….…......36
Table 2.1.1 HCR solutions and buffer recipes compatible to short DNA probe.........42
Table 2.1.2 DNA HCR probe sequences against vegfr1 transcript.............................44
Table 2.1.3 DNA HCR probe sequences against vegfr2 transcript.............................47
Table 2.1.4 DNA HCR probe sequences against vegfr3 transcript.............................50
Table 2.2.1 Basic Filter Sets: confocal acquisition on Zeiss 780 Upright..................56
Table 2.5.1 List of equation defining tissue specific & global Cluster Volume
Occupancy.................................................................................................................76
Chapter 3: Exploring the role of vegfr1, vegfr2 and vegfr3 transcripts during angiogenesis and
vasculogenesis ……………………………………………………….…....................77
Table 3.2.1 t-test scores for tissue specific sCVO.....................................................88
Table 3.2.2 t-test scores for global gCVO................................................................88
Table 3.3.1 t-test scores in sCVO between WT and mutants in anterior vessels........96
Table 3.3.2 t-test scores in sCVO between WT and mutants in all vessels.................96
Chapter 4: Future Directions…………………………………………………………….…......N/A
viii
LIST OF FIGURES
Chapter 1: General Background…………………………………………………………….…......1
Figure 1.1.1 The basis of vasculogenesis; de novo formation of vessels from
angioblasts...................................................................................................................4
Figure 1.1.2 Consecutive stages of vessel sprouting....................................................5
Figure 1.2.1 Vasculogenesis in the zebrafish embryo................................................10
Figure 1.2.2 Sprouting angiogenesis in the zebrafish embryo...................................11
Figure 1.3.1 VEGF receptor binding properties.........................................................16
Figure 1.4.1 Endocytosis regulates VEGFR2 signaling during angiogenesis............21
Figure 1.5.1 Schematic representation of current vs literature results........................29
Figure 1.6.1 HCR multiplexed imaging in fixed zebrafish embryos..........................33
Figure 1.6.2 HCR hairpin mechanism.......................................................................34
Figure 1.6.3 HCR schematic of detection and amplification stage.............................35
Chapter 2: Methods and Techniques……………………………………………………….…......36
Figure 2.1.1 HCR schematic of detection and amplification stage.............................38
Figure 2.2.1 Confocal microscopy apparatus............................................................54
Figure 2.2.2 Imaris stitcher on tile images.................................................................55
Figure 2.3.1 HYSP unmixing for eGFP and Alexa 514 overlapping spectra.............58
Figure 2.3.2 Control signal for hyperspectral unmixing............................................60
Figure 2.4.1 Methods workflow for semi-quantification of transcript clusters..........64
Figure 2.4.2 Hyperspectral Unmixing of multiplex HCR samples............................66
Figure 2.4.3 Imaris volume rendering........................................................................68
Figure 2.4.4 Transcript cluster creation.....................................................................70
Figure 2.4.5 Sample cluster quantification schematic...............................................71
Figure 2.4.6 Identifying nuclear active transcripts and nuclear volume
Fingerprint.................................................................................................................72
Figure 2.5.1 Definition of Cluster Volume Occupancy of vegfr1 in Dorsal
Aorta.........................................................................................................................75
Chapter 3: Exploring the role of vegfr1, vegfr2 and vegfr3 transcripts during angiogenesis and
vasculogenesis ……………………………………………………….…....................77
Figure 3.1.1 Defining the developmental wave of ISVs within the same stage
or along various stages during vascular development................................................79
Figure 3.2.1 vegfr transcripts exhibit granulated and distinct localization
pattern in various vessels during angiogenesis and vasculogenesis...........................84
Figure 3.2.2 Cluster Volume Occupancy of vegfr transcripts at 26hpf, for
multiple embryos.......................................................................................................86
Figure 3.3.1 Comparing the anterior part of zebrafish embryos between WT
and dnm1l mutants at 26hpf.......................................................................................92
Figure 3.3.2 Comparing the posterior part of zebrafish embryos between WT
and dnm1l mutants at 26hpf.......................................................................................93
Figure 3.3.3 Distribution plots quantifying the differences between WT and
dnm1l mutants in the anterior and posterior parts at 26hpf.........................................94
Figure 3.3.4 vegfr1 and vegf3 transcript expression is higher in dnm1l mutants,
ix
while vegfr2 expression is not significantly affected.................................................95
Figure 3.4.1 Expression of vegfr transcripts in posterior part during first and
second wave of angiogenesis in various endothelial tissues....................................100
Figure 3.4.2 Expression of vegfr transcripts in anterior part during first and
second wave of angiogenesis in various endothelial tissues....................................103
Figure 3.4.3 Mean intensity vs cluster volume scatter plots during 1
st
and 2
nd
wave of angiogenesis in whole mount embryos......................................................106
Figure 3.5.1 Nuclear expression fingerprint of vegfr1 and vegfr2 active
transcription on all vessels between Posterior vs Anterior.......................................111
Figure 3.5.2 Nuclear expression fingerprint of vegfr3 and vegfr2 active
transcription on all vessels between Posterior vs Anterior.......................................113
Figure 3.5.3 Nuclear dot transcript expression fingerprint of vegfr2, vegfr2 and
vegfr3 active transcription in posterior vessels........................................................115
Figure 3.5.4 Nuclear dot transcript expression fingerprint of vegfr2, vegfr2 and
vegfr3 active transcription on all vessels between Posterior vs Anterior..................117
Chapter 4: Future Directions…………………………………………………………….….......123
Figures 4.4.1 HCR schematic of detection and amplification stage.........................129
x
ABBREVIATIONS
AF: Auto Fluorescence
CVO: Cluster Volume Occupancy
DA: Dorsal Aorta
DAPI: 4′,6-diamidino-2-phenylindole
DNA: Deoxyribonucleic Acid
Dnm1l: Dynamin 1 like
ECs: Endothelial Cells
eGFP: enhanced Green Fluorescent Protein
en: endoderm
FISH: Fluorescent In Situ Hybridization
flt1: fms related receptor tyrosine kinase 1 (vegfr1 for zebrafish)
flt4: fms related receptor tyrosine kinase 4 (vegfr3 for zebrafish)
HCR: Hybridization Chain Reaction
hpf: Hours Post Fertilization
HYSP: Hyper Spectral software
ISV: Inter Somatic Vessels
kdrl: kinase domain receptor like (vegfr2 for zebrafish)
MBS: Microscope Binocular Stereoscopic
mRNA: messenger Ribonucleic Acid
nc: neural crest
nt: notochord
PBS: Phosphate Buffered Saline
xi
PFA: Paraformaldehyde
PCV: Post Cardinal Vein
PTU: 1-phenyl 2-thiourea
RNA: Ribonucleic Acid
SSC: Saline Sodium Citrate
VEGF: Vascular Endothelial Growth Factor ligand (protein reference)
VEGFR1: Vascular Endothelial Growth Factor Receptor 1 (protein reference)
vegfr1: Vascular Endothelial Growth Factor Receptor 1 (transcript reference)
VEGFR2: Vascular Endothelial Growth Factor Receptor 2 (protein reference)
vegfr2: Vascular Endothelial Growth Factor Receptor 2 (transcript reference)
VEGFR3: Vascular Endothelial Growth Factor Receptor 3 (protein reference)
vegfr3: Vascular Endothelial Growth Factor Receptor 3 (transcript reference)
WT: Wildtype Phenotype
xii
UNITS
AU: Airy Units
a.u.: Arbitrary units
o
C: Celsius (temperature)
dpi: dots per inch
μL: microliters (sample volume)
μm
3
: cubed microns (cluster volume)
μΜ: micro Molar (sample concentration)
nm: nano meters (wavelength)
nM: nano Molar (sample concentration)
pM: pico Molar (sample concentration)
xiii
ABSTRACT
An increased understanding of the molecular mechanisms that controls angiogenesis and
vasculogenesis is critical for the development of novel therapeutic strategies against vascular
diseases. These diseases can re-activate embryonic vascular formation signaling events. An ideal
animal model to study vascular development is zebrafish (Danio rerio) due to its transparency and
easy in vitro/in vivo experimental manipulation. During embryogenesis, blood vessel precursors
undergo a series of complex morphogenesis events and elaborate in vascularization of adult
zebrafish body. A key regulator of vascular development is VEGF receptor 2 (VEGFR2), which
plays a crucial role in all aspects of normal, and pathological vascular endothelial cell dynamics.
Its dynamic expression interconnects with other vascular endothelial growth factor receptors, such
as VEGFR1 & VEGFR3. This PhD thesis aims to elucidate the dynamics of vegfr transcript
localization during angiogenesis of ISVs and vasculogenesis of DA and PCV; how their
cytoplasmic transcript localization can interconnect with receptor endocytosis and patterns of
transcript subcellular localization during 1
st
and 2
nd
wave of angiogenesis. With the use of confocal
microscopy, cutting edge multiplexed in situ hybridization chain reaction (HCR), hyperspectral
signal unmixing (HYSP) and post imaging analysis, a coherent mapping of mRNA expression is
performed within intact whole mount zebrafish embryos. Furthermore, multiplex labelling of
vegfr1, vegfr2 and vegfr3 is allowing a thorough transcript localization analysis in developing
vasculature between 1
st
and 2
nd
wave of angiogenesis.
1
CHAPTER 1 General Background
1.1 Vasculogenesis, angiogenesis: Two important morphological processes
In recent years, the zebrafish (Danio rerio) has risen through experimental procedures as
an excellent vertebrate model system for studying embryo development (Barut et al, 2000) and in
particularly endothelial vascular development (Lawson et al, 2002). The zebrafish is carrying out
the following important anatomical features that make it an ideal candidate : low opacity and high
optical transparency in all embryos and specific adult fish transgenic lines such as Casper line
(White et al, 2008). Furthermore, small size and fast accessible development; easy experimental
manipulation in vivo and in vitro; relative low cost of growing and handling compared to other
animal models; similar genetic structure to humans, sharing ~70% of genes (Kardash, 2012). All
these factors have contributed altogether to make zebrafish (Danio rerio) an ideal candidate for
basic scientific research.
In this PhD Thesis, we will explicitly focus on two fundamental processes during vascular
development: vasculogenesis and angiogenesis. These are two main morphological processes
which are very distinct from each other. Vasculogenesis is defined as the de novo formation of
vessels from mesoderm-derived endothelial precursors (angioblasts) (Swift and Weinstein, 2009),
while angiogenesis is the formation of blood vessels from pre-existing ones. It has been initially
characterized as a sprouting process under which a new vessel is sprouting, extending and
branching from a primary blood vessel (Patan, 2000). In addition to this sprouting mechanism of
angiogenesis, a completely different mechanism exists; intussusception. During this second type,
vessels split into two different branches while having the same vascular base. It is mostly
characterized in mammals (Makanya et al, 2009), thus we will not include it in this current study
of the vertebrate zebrafish model. Amidst of embryonic development of zebrafish (Danio rerio),
2
the first vessels form through vasculogenesis (Figure 1.1.1, based on Potente et al, 2011).
Heterogeneous mesenchymal progenitors are differentiating into angioblasts (Swift and Weinstein,
2009), which are endothelial precursors. Subsequent assembly elaborates a network of newly
formed vascular cords (Adams and Alitalo, 2007). They form a primitive vascular network that
will give rise to a mature endothelial structural network (Potente et al, 2011) differentiated into
arteries and veins.
Artery and vein derived endothelial cells express specific molecular markers and have
district identities (Adams and Alitalo, 2007, Swift and Weinstein, 2009). These expressions
combined with molecular feedback loops of branching pattern generator and molecular signatures,
create a dynamic interplay of important gene expression. Later, we will focus analytically into the
most critical molecular signatures of arterio-venous differentiation and their interconnection with
critical regulators of vascular development downstream of this chapter [for further info see Part
1.5 of Chapter 1]. At this point in order to further introduce the concept of sprouting angiogenesis
in zebrafish, we need to underline that dorsal aorta is the very first structure that forms after the
above described vasculogenic assembly, differentiation and lumen formation.
Following the lumenization of dorsal aorta at 24hpf, the artery derived endothelial cells
(ECs) give rise to secondary structures through the first (1
st
) wave of sprouting angiogenesis. This
important physiological process allows new vessels to form from pre-existing ones that will
elaborate in the developing vasculature (Potente et al, 2011). More specifically in zebrafish,
sprouting angiogenesis is giving rise to the development and scaffold formation of Inter Somitic
Vessels (ISVs). This physiological mechanism resembles new vessel formation during
inflammation or tumor growth in humans (Moshal et al., 2010) [for further info see Part 4.1 of
Chapter 4].
3
During sprouting angiogenesis endothelial cells of the DA are getting activated by
proteolytic breakdown of the basement membrane and their subsequent coat of mural cells that
protect the EC layer. As described in Figure 1.1.2, from (Potente et al, 2011), endothelial cells
(ECs) that are getting activated are beginning to form tip cells with extending filopodia. They
extend dorsally in order to follow VEGF cues and they differentiate into tip and stalk cells (for
anatomical differences refer to Figure 1.2.2) by the Notch pathway (Eilken et al., 2010; Phng et
al, 2009). Tip cells respond to proangiogenic signals and cause them to extend filopodia. As they
sprout and proliferate, they further divide into cells creating a structure of tip and stalk cells. Those
stalk cells that support further the ISV structure, exhibit a dynamic behavior of suppressed
filopodia expression. They further coordinate into different branches, elongate simultaneously in
different locations in order to finally fuse with neighboring ones. At this point, they build vessel
loops that mature and lumenize, allowing initiation of blood flow that continues throughout the
network of newly formed vessels.
These basic concepts of sprouting angiogenesis and vasculogenic assembly will further
guide us into following simultaneously the morphogenic effects taking place in developing
vasculature of the zebrafish embryo. It is important to introduce these concepts in order to further
understand the characteristic molecular signatures of different types of developing vessels that we
will cover in the data section of Chapter 3.
4
Figure 1.1.1: The basis of vasculogenesis; de novo formation of vessels from angioblasts
(picture inspired from Potente et al, 2011). Heterogenous mesenchymal progenitors acquire
different identity specification. Angioblasts give rise to endothelial cells (ECs) expressing markers
that differentiate them from their mesenchymal progenitors. Furthermore, segregation into
important compartments and subsequent assembly give rise to elongated vascular cords. ECs
exhibit various fates of artery and vein differentiation, giving rise to distinct artery and vein
precursor cords. Those precursor cords will further lumenize in order to allow fully developed
blood flow circulation.
5
Figure 1.1.2: Consecutive stages of vessel sprouting (picture inspired from Potente et al, 2011).
Proangiogenic signals activate endothelial tissues and cause them to extend filopodia in order to
sense them. These primitive extensions create new tip cells (green) that follow angiogenic signals.
By sprouting and proliferating, they create cell structures of tip cells (green) expressing filopodia
and stalk cells (yellow) that support elongation. They further coordinate into different branches,
elongate simultaneously in different locations in order to finally fuse with neighboring ones. At
this point, they build vessel loops that mature and lumenize, allowing initiation of blood flow that
continues throughout the network of newly formed vessels.
6
1.2 Vascular morphogenesis and angiogenesis in zebrafish
In this PhD thesis part, we will focus on these two main morphogenic processes and how
they apply throughout different stages of vascular development in zebrafish (Danio rerio). We
need to establish the essential background in understanding vasculogenesis and sprouting
angiogenesis in zebrafish as in the following chapters we will further elucidate the role of critical
molecular signatures and their expression dynamics. Two important waves of sprouting
angiogenesis are taking place between the developmental stages of interest (26-34hpf). Each
sprouting or vessel formation event is deriving from district endothelial regions with diverse
molecular identities (Ellertsdóttir et al, 2010; Potente et al, 2011). It is pertinent to underline the
importance of this study; the mechanism under which developing vasculature forms and assembles
vessels is molecularly similar as in mammals and other higher vertebrates (Isogai et al., 2001).
The first de novo vascular embryonic vessels to appear are dorsal aorta (DA) and post
cardinal vein (PCV) through vasculogenesis (Isogai et al., 2001). During initial zebrafish embryo
developmental stages, vasculogenesis starts at 14hpf with in situ aggregation of angioblasts
towards the midline below the hypochord in two lateral stripes. Those angioblasts will give rise to
endothelial cells and hematopoietic cells (Ellertsdóttir et al, 2010). At 17hpf, angioblasts start
expressing markers of arterio-venous differentiation. At 21hpf, the cells that are located ventrally
of the vascular cord will start migrating just below the forming Dorsal Aorta (DA). DA lumenizes
prior to the Posterior Cardinal Vein (PCV) and Central Vein (CV) in absence of blood flow
(Eriksson et al, 2000; Herbert et al., 2009; Jin et al., 2005). At this stage, venous differentiated
angioblasts will aggregate towards and hollow around the blood cells to ultimately form a vein
tube (Gore et al., 2012; Ellertsdóttir et al, 2010). Around 28-30hpf, the artery and vein are fully
formed, lumenized and can carry blood flow (Roman et al., 2002) as presented in Figure 1.2.1.
7
Furthermore, another important structure will arise from the pre-existing DA and PCV,
following somite generation from anterior to posterior; the Intersegmental Vessels (ISVs). The
ISV formation during zebrafish embryonic development comes in two (2) major waves of
angiogenic sprouting (Isogai et al., 2003). The first wave of sprouting angiogenesis forms the
Segmental Arteries (SA) and the second one the Segmental Vessels (SV) and further the
Parachordal Lymphangioblasts, as presented in Figure 1.2.2. We will focus on the physiological
mechanism governing these morphological changes in the developing embryo, between the stages
22-34hpf.
Sprouting angiogenesis starts at 22hpf on the most anterior part of the trunk area, where
one or two cells sprout out of the DA epithelium . This event marks the initiation of the first (1
st
)
wave of sprouting angiogenesis. It starts on the anterior part of the zebrafish embryo (22hpf) and
follows the somite development (for more info on somite development and segmental clock see
Part 3.1 of Chapter 3). Each ISV will sprout bilaterally and will follow an angiogenic wave from
anterior to posterior that will resume at 26hpf towards the tail. Forming ISVs will follow the somite
boundaries, extend and proliferate in order to grow dorsally towards the neural tube. During the
stages of development, the endothelial sprout consists of 2-4 cells that are stabilized by inter
endothelial junctions. As they proliferate towards the dorsal part of the embryo, they develop into
3-4 dividing cells. The guiding cell is expressing filopodia, extending them following
environmental cues, while the rest of the dividing segmental body is consisted by stalk cells as
presented in Figure 1.2.2 or more analytically in Figure 1.5.1. Stalk cells have suppressed
expression of filopodia and are supporting the elongated ISVs and contribute to vessel lumen
formation (Gore et al.,2012).
8
As sprouting angiogenesis is taking place, and the first endothelial cells sprout out of the
DA epithelium with tip cells guiding the sprout and stalk cells following, the ISVs are extending
towards the dorsal part of the embryo at 28-30hpf, as presented in Figure 1.2.2. At this point, they
create a T shape structure and extending until they reach neighboring T-shaped ISVs .They finally
form the future scaffold for Dorsal Longitudinal Anastomotic Vessel (DLAV) around 30-32hpf.
The second (2
nd
) wave of angiogenesis is taking place around 32hpf and is following the
pre-existing scaffold formed by aorta derived ISVs. Similarly, as the first (1
st
) wave of
angiogenesis, 1-2 cells migrate out of the PCV and are forming a sprout (Yaniv et al., 2006). Like
primary angiogenic sprouting these secondary sprouts generate bilaterally. They exhibit a dynamic
behavior with stochastic elements and alternate in their sprouting formation (Herbert et al, 2009).
These sprouts either connect with the adjacent primary vessels and will give rise to SV or generate
a group of lymphatic cells, called parachordal Lymphangioblasts (Isogai et al., 2003). Blood flow
is fully established after SA, SV and DLAV have been formed and lumenized.
In sprouting angiogenesis from the DA, the cells are driven from a gradient of VEGF-A
ligand (Covassin et al, 2009) which appears dispensable for ISV formation. VEGF-A ligand
concentration profile in the extracellular space drives capillary branching and vessel growth
(Ruhrberg et al., 2002). It is the key molecule that drives tip cell migration and controls
proliferation in stalk cells (Gerhardt et al.,2003). Once VEGF-A ligand binds to VEFGR2 and the
receptor gets activated, endothelial cells initiate a cascade response in which junctional
connections between cells are loosening, allowing division and migration towards the angiogenic
stimulus. The tip cell, creating the scaffold for intersomitic vessel formation, receives the highest
VEGFR2 signal (Ellertsdóttir et al, 2010).
9
In the following parts of this PhD thesis, we will focus mainly on the vasculogenesis of
DA and PCV as well as developmental stages of angiogenic sprouting , formation of Segmental
Arteries (SAs) and Segmental Veins (SV) during first and second wave of angiogenesis. Our study
will include transcriptional signatures of important receptors and their dynamic expression
throughout early vessel formation. With the use of robust in situ hybridization techniques, signal
unmixing analysis and volume segmentation, we will be able to semi-quantify those transcript
signals and their nascent transcript expression.
10
Figure 1.2.1: Vasculogenesis in the zebrafish embryo (picture based on Ellertsdóttir et al, 2010).
Schematic cross sections of the trunk showing the formation of DA and PCV at different stages of
vascular development. (A) During medial migration angioblasts (purple) move from the lateral
plate mesoderm towards the endoderm (en) towards the midline below the hypochord (h). At this
position they aggregate to form the vascular chord. At 14-18hpf angioblasts are expressing markers
of arterio-venous differentiation. (B) At 20hpf arterial differentiated cells (red) are located to the
dorsal portion of the vascular rod and will start giving rise to DA. At 21hpf, vein differentiated
angioblasts (blue) and are located ventrally of the vascular cord will begin migrating ventrally and
accumulate below the forming DA in order to contribute to the formation PCV plexus and CV in
later stages. (C) At 24-26hpf, the DA forms and lumenizes prior to PCV&CV in the absence of
blood flow by cord hollowing. Venous angioblasts aggregate and coalesce around the blood cells
to ultimately form a PCV tube enclosing them. (D) At 30hpf, DA & PCV are fully formed and can
carry blood flow. nt: notochord; nc: neural crest;
11
Figure 1.2.2: Sprouting angiogenesis in the zebrafish embryo
Sprouting angiogenesis that leads to the formation of ISV and DLAV in the trunk (Picture inspired
from Ellertsdóttir et al, 2010). At 22-24hpf, ECs from the DA form sprouts lead their way along
the somite boundaries up to the dorsal roof of the neural tube .At 26-28hpf, tip cells send anterior
and posterior extensions in order to connect with their neighboring cells towards the dorsal site.
At 30-32hpf, ECs establish a scaffold consisting of a vascular chord that it is not lumenized yet
and at 32hpf, a secondary wave of sprouting angiogenesis emerges from the PCV connecting with
the adjacent primary intersomitic vessel, which will become segmental vein. Meantime tip cells
fuse on the dorsal part creating a scaffold for DLAV. At 34-36hpf, intersomitic vessels lumenize
allowing blood flow to place in SA, SV, and DLAV.
12
1.3 Critical Regulators during Vascular Development
The main regulators of vascular development during embryogenesis are vascular
endothelial growth factors (VEGFs), which play a catalytic role as well during blood vessel
formation in the adult. There are five major VEGF ligands and they bind with three types of
receptors, vascular endothelial growth factor receptors (VEGFR1,VEGFR2,VEGFR3) and co-
receptors (see Figure 1.3.1) listing all types of angiogenic regulators (Ferrara et al., 2003). The
VEGFRs belong to the RTK superfamily and they belong to the same subclass as receptors for
PDGFs and fibroblast growth factors (FGFs). The extracellular part of VEGFR is around 750
amino acids and it is organized into seven immunoglobulin (Ig-like) folds. The extracellular
domain is followed by; a transmembrane region, a juxta-membrane domain, a split tyrosine-kinase
domain and a C-terminal tail (Keyt et al, 1996). A 70-amino-acid kinase insert interrupts the split
tyrosine-kinase domain.
In PhD thesis work, we concentrate on those three types of receptors and specifically in
their mRNA transcript expression. In agreement to zebrafish nomenclature (Bussmann et al, 2008)
we will provide the alternative names of those receptors. VEGFR2 receptor is equivalent to the
gene names of kinase insert domain receptor like (kdrl) in zebrafish or fetal liver kinase-1 (flk1).
VEGFR1 receptor has alternative name fms related receptor tyrosine kinase 1 (flt1), while
VEGFR3 receptor’s alternative name is fms related receptor tyrosine kinase 1 (flt4).
A wide variety of signaling molecules, receptors and transcription factors have been
dynamically enmesh in the vascularization of zebrafish via regulation, endothelial cell
proliferation and differentiation (Herbert et al, 2011). One of the most critical signaling molecules
for vasculogenesis and sprouting angiogenesis are the endothelial growth factors (VEGFR) and
their receptors (Ferrara et al, 2003; Tammela et al.2005; Roy et al.,2006). Surrounding tissues
13
secreting angiogenic signals produce a wave of VEGF ligands that bind in an overlapping pattern
to three (3) receptors of the tyrosine kinases family (RTKs) on the surface of the cell membrane
as presented in Figure 1.3.1. Receptor-ligand binding is followed by a dimerization
(homodimerization or heterodimerization) of the receptors and autophosphorylation of the tyrosine
kinase domains. Finally, after the event of phosphorylation of the receptor complexes, a wide
variety of downstream cascades is signaling pivotal processes such as sprouting, elongation, cell
proliferation and vascularization; all important processes in zebrafish vascular development
(Lohela et al, 2009).
Multiple vascular endothelial growth factors (VEGFs) exhibit a capacity to bind in more
than one VEGFR receptor dimers; VEGFR-A and VEGFR-C (Takahashi et al., 2005). This ability
of ligand-receptor complex formation gives a versatile dynamic behavior of receptor
phosphorylation cascades activated during vasculogenesis and angiogenesis. More specifically,
VEGF-A ligand can bind in VEGFR2 or VEGFR1 homodimers or VEGFR1/2 heterodimer.
Similarly, VEGF-C ligand can bind both in VEGFR2 or VEGFR3 homodimers or VEGFR2/3
heterodimer in humans (Eriksson et al., 1999; Baldwin et al., 2001). It is apparent that this interplay
of ligand binding creates pairs or receptors competing for dynamic binding of the same ligands.
This unique double binding receptor capacity creates a dynamic environment, where
VEGF-A drives angiogenic sprouting by binding to VEGFR2 but simultaneously VEGFR1 acts
as a sink that limits the concentration gradient and filopodia formation (Habeck et al, 2002). During
development or in tumors, potential blockage of components of Notch signaling, which is normally
upregulated in stalk cells with suppressed filopodia formation, increases excessive tip formation
and filopodia expressions (Thurston et al., 2007). These a dynamic co-operation between Notch
14
signaling, its ligand DLL4, VEGFR1 and VEGFR2 receptor that we will extensively present in
Part 1.5 of Chapter 1.
Important to note that VEGFR2 binds as well to two (2) different ligands (VEGFR-A and
VEGFR-E) adding an extra layer of dynamic behavior. VEGF-A stimulates induction of tip cells
and filopodia formation through VEGFR2, whereas sprouting effects are apparent when VEGFR2
blockage is induced (Phnr et al., 2009).
Similarly, VEGFR3 receptor binds to VEGF-C which is essential for sprouting of LECs in
order to form the trunk lymphatic network, but its loss can be compensated by VEGF-D ligand
(Astin et al, 2014) in mice. VEGFR3 is expressed in endothelial cells but becomes significantly
upregulated in venous derived endothelial cells (Kaipainen et al., 1995). Homozygote inactivation
of VEGFR3 causes lethality during early embryogenesis due to defective vascular development,
while heterozygote mice exhibit severe lymphatic defects (Karkkainen et al.,2000). The Notch
dependent upregulation of VEGFR3 allows vessel formation even in the absence of VEGF-A/
VEGFR2 signaling (Benedito et al., 2012). In zebrafish, VEGF-C expression can be found near
ECs expressing VEGFR3 during lymphangiogenesis. By limiting the abundance of VEGF-C, we
observe failure to establish blood circulation approximating the phenotype in VEGFR3 deficient
mice (Ober et al, 2004).
Apart from lymphatic development, VEGFR3 is associated as well with vein derived EC
network formation and their sprouting during second wave of angiogenesis in zebrafish from
venous derived ECs (Hogan et al., 2009b). In VEGFR3 zebrafish mutants first wave of
angiogenesis can take place independent of VEGFR3 absence, but major defects are apparent
during second wave of venous derived angiogenesis which are identical to VEGF-C morphants
(Hogan et al., 2009a).
15
We conclude that all above-described endothelial growth factor receptors (VEGFRs) are
critical for vasculogenesis and sprouting angiogenesis. Those receptor-ligand binding interaction
combinations are associated with all aspects of normal and pathological vascular endothelial cell
dynamics (Olsson et al, 2006). In the following Part 1.5 of Chapter 1, we will explore the receptors
in the context of Notch signaling, specific arterial venous markers and known traditional in situ
transcript signatures . With the use of HCR, a novel and robust in situ hybridization technique we
will be able to elucidate the transcript expression of those receptors with high signal to noise ratio,
as it has never been reported before.
16
Figure 1.3.1: VEGF receptor binding properties
All above mammalian vascular endothelial growth factors (VEGFs) bind to three kinds of VEGF
receptor (VEGFR) tyrosine kinases, leading to five combinations of receptor dimer complexes.
VEGFRs are formed by homodimers as well as heterodimers. In total we have ten (10) different
combinations of ligand-receptor complexes. Two vascular growth factor ligands are known to bind
in more than one receptor combination, creating a dynamic behavior between pairs limiting ligand
abundance to each other. VEGF-A has the capacity to bind with VEGFR2 as well as VEGFR1 and
VEGFR1/2, while VEGF-C can bind to VEGFR3 and VEGFR2 or VEGFR2/3 heterodimer. The
remaining ligands bind as follows; PDGF and VEGF-B to VEGFR1 only; VEGF-E to VEGFR2 only;
VEGF-D to VEGFR3 only
17
1.4 VEGFR2 receptor endocytosis during angiogenesis
The activity of many ligand-activated transmembrane receptors depends on their
internalization and further trafficking within the cell. The intensity, specificity and duration of the
signal depends whether the receptor will be targeted for degradation or recycling to the plasma
membrane (Gaengel et al, 2013). Right after receptor internalization during endocytosis, many
receptor/ligand complexes continue signaling from endosomal compartments and specific
signaling events occur at district types of endosomes. Endosomes may also serve as scaffolds that
facilitate the assembly of signaling complexes and their subsequent transport to relevant locations
within the cell (Sadowski et al, 2009). All VEGFR receptors that we reviewed in Part 1.3 of
Chapter 1 are ligand activated and their function depends into their subsequent endocytosis, but
we will focus particularly into VEGFR2.
The endocytosis of VEGFR2 is very important for its normal biological function, signaling
capability to facilitate vasculogenesis and sprouting angiogenesis of ISVs. Tip cells expressing
VEGFR2 receptor on their surface follow a flux of VEGF-A ligand expression. Right after VEGF-
A is binding to VEGFR2, the receptor-ligand complex is rapidly internalized in a clathrin and
dynamin dependent manner, ultimately leading towards proteolytic degradation (Lampugnani et
al, 2006). Right after the clathrin-coated vesicle formation, the next step on the endocytic pathway
is the early endosomes. They are located towards the periphery of the cell. Their acidic pH
facilitates the unbinding of ligands from the internalized receptors. Early endosomes contain
multiple tubular extensions that start to form intraluminal vesicles and finally convert into late
endosomes during endosomal maturation.
The basic endocytic pathway has the following elements: a recycling circuit for the plasma
membrane components and their ligands, a macromolecule degradative system and a connecting
18
unidirectional feeder pathway for transport of fluid and selected membrane components from the
recycling circuit to the degradative system . This unidirectional feeder pathway is mediated by late
endosomes, which function as a system for mediating transport of lysosomal components from the
trans-Golgi network to lysosomes. It is not currently clear how transcript dynamics of receptors
interconnect with VEGFR2 endocytosis and if a subcellular machinery exists that combines these
two distinct Rab protein-mRNA receptor transcripts components. In fruit fly embryos (drosophila
melanogaster) it was previously described that bicoid RNA localization requires specific binding
of an endosomal sorting complex (Irion et al., 2007).
The Rab GTPases are key regulatory factors that affect all the above elements of the
endocytic pathway. Specific kinds of Rabs are physically associated with different organelles
during endocytosis as well as their associated transport vesicles (Hutagalung et al, 2011). During
the first stages of endocytosis, the most common small GTPase is rab5 that functions as a
regulatory factor during the early endocytic pathway (Bucci et al.,1992). Rab5 follows the
endocytic membrane from the beginning through various stages of early endosome maturation and
serves as the key regulator of their conversion and maturation to late endosomes (Huotari et al.,
2011). Recently, it was shown that another intermediate component sec14l3 physically binds to
VEGFR2 receptor, prevents its phosphorylation and interacts with rab5a and rab4a on the early
endosomes (Gong et al., 2019).
On the other hand, Rab 7 localizes to late endosomes. It is shown to be important in the
late endocytic pathway functioning as a key regulator in trafficking to lysosomes and governs
early-to-late maturation of endosomes. In addition, Rab9 is also a late endosomal marker, but it
facilitates transport between late endosomes and the trans Golgi network (Lombardi et al., 1993).
Its depletion does not perturb endosome-to-lysosome transport but alters the size and number of
19
multilamellar and dense-tubule-containing structures in late endosomes, as shown in cell cultures
(Gangley et al.2004). Finally, Rab11 is known to associate with perinuclear recycling endosomes
and it is the main GTPase that regulates the recycling of endocytosed proteins at the plasma
membrane (Takahashi et al.2012). It is also required for transport between the trans-Golgi
network-to plasma membrane, recycling endosomes (Chen et al.1998) and it is required for
efficient transport from early endosomes to trans Golgi network (Wilcke et al.2000).
Taking everything into account, endocytosis is the main cellular mechanism that is
responsible for membrane receptor internalization and transportation of ligands or signaling
molecules into the cell. In terms of VEGFR2 receptor perspective described in Figure 1.4.1,
endocytosis starts once a clathrin-coated pit is formed around the VEGFR2/VEGF-A complex.
With the use of dynamin, a mature pit is formed and detach from the plasma membrane towards
the subcellular compartment of the endothelial cell. The newly form clathrin coated vesicle fuses
to an early endosome, where its acidic environment causes the ligand to unbind from the receptor.
Rab 5 functions as a regulatory factor in the early endosomal pathway. At this point VEGFR2 is
either recycled to the plasma membrane and subsequently driven to recycling endosomes for re-
use or will be sorted for degradation into lysosomes with use of late endosomes. In terms of
VEGFR2 receptor recycling, Rab11 is associated with recycling endosomes by regulating the
recycling of endocytosed proteins at the plasma membrane. It is also required for efficient transport
from early endosomes to trans Golgi network. Finally, once the VEGFR2 receptor has been
internalized multiple times and is no longer functional will be transported for degradation in
lysosomes through late endosomes. The transport from late endosomes to lysosomes in
unidirectional, since their fusion is co-dependent. Rab 7 localizes to late endosomes and governs
early-to-late endosome maturation and regulating trafficking towards the lysosomes. Rab9 is a late
20
endosomal marker, but not directly associated with lysosomes; it facilitates transport with the trans
Golgi network. All the above components constitute the main steps of VEGFR2 endocytosis in
endothelial cells and their proper function allows the appropriate sprouting, elongation and
proliferation of activated ECs in ISVs.
In Chapter 3 of this PhD thesis we will concentrate on the phenotype of zebrafish embryos
with mutated function of dynamin-1 like protein; one of the most critical components in the initial
formation of clathrin-mediated endocytosis pits (Pucardyl et al.,2009; Ford et al, 2011),
internalization of caveolae (Henley et al, 1998) and plays as well an important role in the formation
of transport vesicles from the trans-Golgi network (Jones et al., 1998). In zebrafish, dynamin-1
like protein (dnm1l) is one type of dynamin of a large GTPase associated in a wide variety of cell
and organelle fission events. Traditional dynamins are major components of clathrin-mediated
endocytosis and they play a key role into the release of newly formed endosomes (Pucardyl et
al.,2009; Ford et al, 2011). With the use of Gene Trap technology (reference) we integrated a
citrine part of the 10
th
intron (between exon 10-11) of the dnm1l gene , creating a transgenic line
that exhibits a mild and severe phenotype in maternal-derived mutant dnml1 embryos. Mutant
embryos with mild phenotype in endothelial tissues were sorted and used in order to study
subcellular vegfr transcript localization in a disrupted endocytic pathway context (for insights see
results in Part 3.3 of Chapter 3).
21
Figure 1.4.1: Endocytosis regulates VEGFR2 signaling during angiogenesis
Endocytosis starts once a clathrin-coated pit is formed around the VEGFR2/VEGF-A complex.
With the use of dynamin-1, a mature pit is detached from the plasma membrane. The new clathrin
coated vesicle fuses to an early endosome with the use of Rab5, where its acidic environment
causes the ligand to unbind from the receptor. VEGFR2 is either recycled to the plasma membrane
with the use of Rab 11 and subsequently driven to recycling endosomes for re-use or will be sorted
for degradation into late endosomes. Rab11 is also required for transport from early endosomes to
trans Golgi network. Connection from late endosomes to lysosomes in unidirectional, based on
their co-dependent fusion. Rab 7 localizes to late endosomes, governs early-to-late endosome
maturation and regulates trafficking towards the lysosomes. Rab9 is a late endosomal marker, but
not associated with lysosomes; it facilitates transport with the trans Golgi network.
22
1.5 Known association patterns of VEGFRs (type 1,2,3) with Notch
signaling and regulation of vessel morphogenesis in zebrafish
In the following parts of this PhD thesis, we will concentrate on the transcriptional
signatures of the most important vascular endothelial growth factor receptors; vegfr1,vegfr2 and
vegfr3. Before we introduce existing literature referring to those important mRNA transcripts and
their corresponding functional experiments, we need to introduce basic molecular markers and
pathways. One major signaling pathway that is directly correlated with the expression of VEGFRs,
is the Notch pathway which interacts with VEGF ligands. We will focus in markers of
arteriovenous differentiation in vasculogenesis, important signaling pathways in sprouting
angiogenesis and how they interconnect with VEGFR1, VEGFR2 and VEGFR3 receptor
regulation and expression. Further, we will examine in situ transcript labelling in the literature and
known phenotypes associated with vegfr1,vegfr2 and vegfr3 expression.
In previous parts of this Chapter, in terms of vasculogenesis, we referred into markers of
arteriovenous differentiation that start expressing in medial migration angioblasts that move from
the lateral plate mesoderm towards the endoderm of the midline below the hypochord at 14-18hpf.
Arterial and venous endothelial precursors express specific molecular identities (Adams et al,
2007; Swift et al., 2009) that are directly correlated with Notch signaling pathway. Notch pathway
components are highly expressed in arteries but have low levels in veins. In the case of potential
Notch disruption, arteries lose their arterial signature and regain a venous profile differentiation.
Thus, we conclude that Notch upregulation promotes arterial specification and is suppressing
venous identify (Gridley, 2010; Swift et al.,2009).
Notch pathway controls Eph-Ephrin family members which define arterial and venous
boundaries. Ephrin-B2, a member of the ephrin family ligands, increases as Notch is upregulated
23
in arteries and it is absent in veins (Adams et al., 1999; Gore et al, 2012). Contrary, Ephrin-B2
receptor EphB4 expresses in veins following a Notch downregulation. In zebrafish, signals
components act upstream of Notch and upregulate VEGFR that increases Notch components
(Potente et al., 2011). It is clear at this point that potential upregulation of Notch favors arterial
identity during vasculogenesis, while repression of Notch signaling favors venous identity (Swift
et al, 2009). Targeted gene deletion of Ephrin-B2 ligand or its receptor EphB4 resulted in similar
vascular development abnormalities, suggesting that they directly interact with one another as
regulators of cell contact dependent signaling (Pitulescu et al.,2010)
In terms of sprouting angiogenesis, tip cells are sprouting from DA during first (1
st
) wave
of angiogenesis. As mentioned previously, they extend dorsally in order to follow VEGF cues and
they differentiate into tip and stalk cells (for anatomical differences refer to Figure 1.5.1) by the
Notch pathway (Eilken et al., 2010; Phng et al, 2009). Extensive analysis of Notch signaling
pathway reveals low Notch activity in tip cells, which exhibit high filopodia expression. In stalk
cells, which are ECs that express several Notch receptors, Notch1 is critical for subdue tip cell
behavior thus creating significantly suppressed filopodia extensions (Potente et al., 2011). Tip cells
express upregulation of DLL4 component, which is a Notch ligand. During development in
tumors, blockage of Notch signaling pathway or DLL4 increases filopodia extensions and
sprouting (Thurston et al., 2007). This characteristic behavior of hyper sprouting and excessive
number of tip cell heaving ECs following Notch inhibition indicate that tip cell phenotype is the
default endothelial response to proangiogenic signals (Potente et al., 2011).
Understanding the basic profiling of Notch signaling upregulation in stalk cells and artery
derived endothelial cells creates a dynamic picture of how sprouting angiogenesis and
vasculogenesis interconnect with various types of VEGF ligands and expression of VEGFR1,
24
VEGFR2 and VEGFR3 receptors. From the beginning of heterogenous mesenchymal progenitors
differentiating to angioblasts and migrating towards the endoderm the VEGFR2 expression is
apparent. Few of these migrating angioblasts will exhibit early expression of VEGFR2, a marker
that will subsequently define the endothelial population (Gering et al, 1998). VEGF-A, a ligand
that drives the expression of VEGFR2 in angioblasts, leads to the activation of Notch signaling
which results in arterial differentiation of a subset of angioblasts the Potential loss of VEGFR2
function causes defects in endothelial cell development (Gore et al, 2012; Covassin et al.,2009).
Particularly, various mutations in the zebrafish VEGFR2 receptor are causing apparent
dorsa aorta (DA) and partial segmental artery (SA) defects with differential phenotype; different
mutations on the VEGFR2 (kdrl) gene exhibit divergent phenotypes during vessel morphogenesis
(Covassin et al., 2009). Most severe phenotypes involve various acute defects on segmental artery
(SA) formation and small filopodia extensions on them, while in some cases sprouts failed to
extend properly from the DA. Furthermore, mutant zebrafish embryos failed to have a separated
DA and PCV formation. Boundaries between artery and vein differentiated ECs were not clear and
instead they appeared as a uniform major trunk vessel.
Similar vascular remodeling defects have been reported when there is loss of Ephrin-B2
ligand and its EphB4 receptor; two major regulators of cell-contact dependent signaling (Pitulescu
et al., 2010). As we have mentioned before Eph-Ephrin binding leads to bidirectional signaling in
cells expressing the ligand Ephrin B2 (Potente et al., 2011) such as arteries or stalk cells (reverse
signaling) or expressing the receptor EphB4 such as veins or tip cells (forward signaling).
Endothelial cells (ECs), such as arteries or stalk cells, that are lacking Ephrin B2 are unable to
properly internalize VEGFR2 and VEGFR3. This defective endocytosis on ECs that actively need
25
to transmit properly VEGFR-A or VEGF-C signals leads into impairment of angiogenic sprouting
(Sawamiphak et al., 2010, Wang et al., 2010).
By reviewing the existing literature described above in terms of functional expression of
VEGFR2 receptor that expresses in all EC progenitors we conclude that this unique receptor is a
key component of embryonic vascular development (Millauer et al, 1993). Thus, we are expecting
a universal expression of its mRNA transcript in all ECs. Traditional in situ hybridization analysis
of vegfr2 (kdrl) reveal early expression during vasculogenic assembly at the DA (18hpf), strong
expression in DA and sprouting ISVs, but weaker in PCV (22-28hpf) and finally strong expression
in the same vessels but absent in DLAV (Shane et al., 2012)
In addition, with expression of VEGFR2 in endothelial cells, analyses in mice models have
revealed an additional molecular mechanism that limits the number of sprouts; the VEGFR1 (FLT-
1) receptor. This receptor is secreted from stalk cells adjacent to the forming sprout (Chappell et
al., 2009). As we already have reviewed in part 1.3 of Chapter 1, VEGF-A ligand binds as well to
VEGFR1 homodimers and VEGFR1/2 heterodimers, thereby limiting it from the environment
surrounding tissue around the angiogenic sprout. VEGFR1 has two major roles in sprouting
angiogenesis; it regulates the proper formation of the filopodia extensions in activated ECs or
sprouts by acting as a sink for surrounding VEGF-A, guiding tip cells towards one direction and
preventing them to connect back to its original position (Ellertsdóttir et al, 2010).
In zebrafish, VEGFR1 acts in a Notch-dependent manner as a negative regulator of tip cell
development and branching patterns in sprouting angiogenesis (Krueger et al., 2011). In case of
VEGFR1 morphants, there is an excessive tip cell formation and filopodia extensions, an
increased number of ECs in ISVs comparing to wildtype and overexpression of sprouting behavior.
Over expression of VEGFR1 results in short artery sprouts and limitation of tip cell behavior. This
26
morphant phenotype is similar with the effects of Notch signaling loss (Siekmann et al, 2007)
observed in tip cells of wildtype embryos. In stalk cells, which are ECs that normally express
several Notch receptors, Notch1 is critical for subdue tip cell behavior thus creating significantly
suppressed filopodia extensions (Potente et al., 2011). In terms of following vegfr1 transcript
expression (Krueger et al., 2011), soluble and membrane bound forms of VEGFR1 were used and
identified as following with traditional in situ hybridization between 24-48hpf ; mVEGFR1 was
expressed in DA, PCV and ISVs while sVEGFR1 was specifically expressed in DA and ISVs but
not in PCV.
In contrast to arterial development and sprouting angiogenesis which is dependent to
VEGFR-A signaling and active dynamic competition between VEGFR2 and VEGFR1 receptors ,
venous fate of active ECs follows VEGF-C ligand (Covassin et al., 2006; Lawson et al., 2003). In
zebrafish, the main receptor that plays a key role in vasculogenesis and lymphangiogenesis is the
VEGFR3 (FLT4) signaling. Along with its corresponding ligand VEGF-C, there are necessary
components of venous sprouting in second (2
nd
) wave of angiogenesis (Hogan et al, 2009). In terms
of early vasculogenesis, those concepts can be verified as the negative modification of VEGF-A
or Notch signaling levels influences the angioblast migration, segregation and ventral sprouting
behavior of vein derived ECs. If Notch signals were blocked or VEGF-A expression was inhibited,
then angioblasts showed excessive ventral migration. Also downregulation of VEGFR3 led to a
reduction in ventral sprouting (Herbert et al., 2009).
Furthermore, in terms of VEGFR3 ( flt4) the receptor was believed until recently to solely
express in venous derived endothelial cells (Hogan et al., 2009b) of vertebrates (Hogan et al.,
2009b) however it has recently identified in arterial cells as well (Covassin et al., 2009; Siekmann
and Lawson, 2007). VEGF-C is essential on the aspect of venous angiogenic sprouting (Hogan et
27
al.,2009b). There is a high abundance of VEGF-C ligand in the developing embryo, especially in
the trunk, initially at the hypochord and later in the DA, during both angiogenic waves (Covassin
et al., 2006). Same applies as well to VEGFR3 receptor expression as it exists in endothelial cells
during both waves of sprouting angiogenesis. It has been an open question how developing arteries
and veins have been programmed to uniquely respond to VEGFR3/VEGF-C signaling. In order to
address this question, we have previously expanded on the role of Notch signaling suppression in
vein derived ECs and how it interconnects with VEGFR3 upregulation.
Previous studies in zebrafish have shown that potential disruption of intracellular Notch
signaling (mind bomb mutants), which translates into a uniform impairment of Notch signaling,
exhibits an upregulated VEGFR3 expression in arterial derived endothelial cells throughout
development (Lawson et al.2001). DLL4, a protein that binds to a Notch1 receptor, provides an
essential mechanism of the Notch signaling pathway that suppressed the response to VEGF-C
induced signaling of VEGFR3 expression throughout development in arteries. When this protein
component is lost , we observe severe hyperbranching due to VEGF-C and VEGFR3 receptor
activity in the DA (Hogan et., 2009).
We introduced this concept of DLL4 expression and upregulation of Notch signaling in
order to put into context the VEGFR3 transcript levels in the developing arteries after the first
wave of angiogenesis. It has been found that vegfr3 transcript levels are progressively lowered in
developing DA and ISA after 24hpf. This explains why in zebrafish the vegfr3 transcript exists
mostly in PCV and slightly in stalk cells of ISAs at 26hpf. Interestingly, when DLL4 action is
impaired although arteries become more responsive to VEGF-C or VEGFR3, their transcription
levels do not change during first wave of angiogenesis, suggesting that some unknown molecular
regulator is involved in the gene expression of vegfr3, vegfc or vegfd.
28
In this part of the PhD chapter, major findings from existing literature are covered, which
are referring to VEGFR1,VEGFR2 and VEGFR3 receptors. Their activity interconnects with the
up- or downregulation of the Notch signaling pathway, and it is the phenotype of their morphants.
We found many transcript expressions patterns of those receptors (vegfr1, vegfr2 and vegfr3) and
we can later on understand them in terms of different Notch expression levels of various vessel
types. In general, when looking into traditional in situ staining results or transcript level analyses,
our main conclusions show that; vegfr2 is expected to be uniformly expressed in all vessel types;
vegfr1 transcript expression would be limited to artery derived ECs; vegfr3 is expected to be
present in vein derived EC. In Chapter 3, with the use of a high signal-to-background in situ
hybridization chain reaction (HCR), novel signal unmixing techniques , that allow semi
quantitative imaging approaches we will explore their localization patterns. During primary and
secondary angiogenic sprouting we will explore the transcript signatures of those important
receptor transcripts throughout vascular development in zebrafish.
29
Figure 1.5.1: Schematic representation of current vs literature results
Comparing results of transcript localization after HCR in situ staining, according to present
transcript data presented in Chapter 3 versus traditional chromogenic in situ hybridization staining
from existing literature (Herbert et al., 2012, Habeck et al., 2002)
30
1.6 Introduction to in situ Hybridization Chain Reaction (HCR)
In situ hybridization methods allow imaging of mRNA expression in a spatial and
morphological context that ranges from sub-cellular resolution to whole mount organism levels.
Traditional in situ hybridization (ISH) techniques allow mRNA labelling in whole mount zebrafish
embryos with the use of NBT (Nitro Blue Tetrazolium) oxidization (Heiles et al., 1988), but have
limited resolution due to staining capacity. Other, fluorescent in situ techniques (FISH) allow for
spatiotemporal resolution but are limited due high background in case of low signal expression
levels (Huber et al., 2018). In order to address the classical labelling limitations of traditional FISH
techniques, a novel concept of in situ hybridization chain reaction (HCR) has been introduced.
HCR allows multiplex mRNA labeling with the use orthogonal HCR amplifiers that can operate
simultaneously within the same sample and allow high signal-to-background ratios (Choi et
al.2010).
An HCR amplifier is composed by two unique nucleic acid hairpin species (H1 and H2 as
Figure 1.6.2) that are specifically designed in order to co-exist metastably in hairpin loop form, in
the absence of their corresponding nucleic acid initiators. Each HCR hairpin consists of two major
parts; an input domain that has an exposed single stranded sequence (Initiator complementary
sequence) and an output domain with a single stranded toehold sequence (hairpin loop). Each
hairpin is fluorescently labeled at the end of the single strand, opposite of the position of the
complementary initiator sequence. Only in the presence of a probe initiator the exposed single
strand of the input hairpin domain of H1 hybridizes, causing the hairpin to open and expose its
previously toehold sequence of the output domain. After annealing, the hybridization of H1 output
domain to the input domain of H2 opens in turn the hairpin loop of output domain in H2. Then as
H2 unfolds is exposing its output domain that is identical to the probe initiator sequence
31
(mechanism described in Figure 1.6.2). This provides the basis for a chain reaction of alternating
H1 and H2 sequences that polymerize in order to form a tethered amplification DNA polymer that
has multiple fluorescent labels (mechanism described in Figure 1.6.3).
The HCR technique is consisted of two major labeling stages: the detection stage and
amplification stage, each having a 12-16h incubation period. During the detection stages, multiple
probes recognizing different mRNA parts are added into the sample. Each probe has a single
stranded part that is extending out of the double stranded structure. This single stranded part is the
Initiator of H1 hairpin system used. Excessive probes are sequentially washed out of the sample,
prior to signal amplification. During amplification stage, metastable hairpins H1 and H2 are added
into the sample. They created a double stranded DNA polymer that extends from each initial probe
position on the mRNA. Hairpins in excess are washed out, allowing bound hairpin polymers to
exist (mechanism described in Figure 1.6.3).
This programmable in situ amplification mechanism has some important in situ detection
properties that favor simultaneous multiplexed amplifications. Firstly, the programmable
chemistry of this nucleic acid technology allows the creation of multiple mRNA probing sequences
to exist with different initiator systems . This variation of nucleic acid technology allows multiplex
orthogonal transcript labeling that operate independently within the same sample (systems B2-B4
shown on figure 1.6.3) with the use of their corresponding hairpins. Secondly, it is an isothermal
triggered self-assembly process that allows user friendly and uniform detection/amplification in
whole mount embryos. Finally, it allows high labeling sensitivity with a high signal-to-background
ratios capacity that enables high spatial sample resolution under confocal microscopy conditions.
HCR is an exceptional tool that allows us to study multiple targets on the same time (as
presented in Figure 1.6.1) and can even further be evaluated in a semi-quantitative manner. In the
32
following chapters we will use this powerful in situ hybridization technique in order to study the
transcript signatures of vegfr1, vegfr2 and vegfr3 receptors during vasculogenesis, the primary and
secondary sprouting angiogenesis. Combined with hyperspectral imaging methods and novel
signal unmixing techniques, we would be able to estimate spatiotemporal expression and semi-
quantify the transcript expression levels.
33
Figure 1.6.1: HCR multiplexed imaging in fixed zebrafish embryos
(A) Schematic expression of three (3) distinct mRNAs in lateral view; kdrl (red), citrine (green),
tpm3 (blue). (B-C) With the use of confocal microscopy, mRNA expression is mapped at multiple
z positions. With the use of HCR detection, three different orthogonal initiator systems were used
(B2,B3,B4). HCR amplifiers carry spectrally different fluorophores (Alexa 488,647 and 594).
Maximum intensity projection at lateral view of x-y axis (B) and within a 30μm embryo section
(C). Expression of transcripts at z=0μm, only tpm3 is visible in the muscle tissue(D), at z=10 and
z=22μm citrine probes are labeling the eGFP expression on ECs and kdrl the transcript of the
VEGFR2 receptor (E-F). Scale bar is at 10um.
34
Figure 1.6.2: HCR hairpin mechanism (Picture based on Choi et al, 2010).
Metastable fluorescent DNA hairpins initially exist into separate solutions prohibiting self-
assembly. Upon addition of the Initiator Type I (red), the Gibbs free energy of the metastable
hairpin system changes allowing annealing of the hairpin structure H1 (light green). At this point
the initiator triggers a chain reaction, as part of annealed hairpin H1 causes simultaneous annealing
of hairpin H2 (dark green). Each time a hairpin anneals in alternate terms, they self-assemble into
fluorescent amplification chain reaction of alternative H1-H2. Those polymerization steps amplify
the detection signal of a specific initiator system. Fluorophores are labeled with magenta stars.
35
Figure 1.6.3: HCR schematic of detection and amplification stage (Picture based on Choi et al,
2010). Detection Stage: Probe sets with initiator on the 5’ part hybridize to mRNA target
transcripts. Unbound probes are washed away. Amplification Stage: Initiators are snapped cooled
and added to the sample. As they consecutively anneal, then they trigger a chain reaction and
create a self-assembly of tethered fluorescent amplification polymers. Excessive hairpins are
immediately washed away from the sample. Experimental timeline, of an example of three (3)
different mRNA targets that are amplified simultaneously. Multiplex HCR labeling is possible
since different types of initiators trigger orthogonal HCR amplification cascades of corresponding
hairpins. Each set of initiators (System B2,B3,B4) has its corresponding H1-H2 hairpin system
that contains distinct fluorophores. Incubation times for detection and amplification stage lasts
16hours each.
36
CHAPTER 2 Methods and Techniques
2.1 In situ Hybridization Chain Reaction Methods and Protocol
For multiplex orthogonal labelling of three (3) transcript targets vegfr1, vegfr2 and vegfr3,
a customized mRNA labelling system has been used in combination with commercial hairpins
(Alexa 514-B2, Alexa 594-B3, Alexa 647-B4) from Molecular Instruments, Inc. Probes where
designed with specialized DNA probe design software (Stellaris Probe Designer, LGC Biosearch
Technologies) and extended to 30nt probe sequences according to their original gene coding
sequence (SnapGene) and optimized melting temperatures. Furthermore, by using a customized
set of probes (Integrated DNA Technologies, Inc.), we optimized the original HCR protocol (Choi
et al., 2010), which was utilizing instead 50nt long probe sequences for target transcript labelling.
Instead of previously reported two (2) initiator Systems we used only one (1) on the 3’prime end
of all probe sequences.
Zebrafish embryos were collected at 26-34hpf depending on the developmental stage of
interest and transgenic expression, incubated at 28
o
C with phenylthiourea (PTU) egg water
solution and dechorinated prior to fixation. For fixation of whole mount zebrafish embryos, freshly
made 4% paraformaldehyde (PFA) solution was used by diluting 32% PFA stock into 1X
phosphate-buffered saline (PBS). Fresh PFA aliquots lower the levels of embryos autofluorescence
in downstream imaging conditions.
Finally, to minimize developmental variability within sample batches in experiments
where semi-quantitative data analysis was needed, a large pool of embryos was incubated at 28
o
C
and subsets of embryos were fixed every 2hour of various developmental stages. All embryos were
fixed with same 4% PFA master mix solution to avoid potential intrinsic autofluorescence signal.
All embryos were simultaneously treated with the following HCR Protocol to avoid variation in
37
detection and amplification stages for ensured reproducibility and downstream semi-quantification
conditions.
The HCR protocol is described below; it includes analytical steps during Preparation,
Detection and Amplification stage. All buffers are described in Table 2.1.1 and specifically
designed DNA probes against vefr1, vegfr2 and vegf3 transcripts are sited in Table 2.1.2, Table
2.1.3 and Table 2.1.4 respectively.
38
Figure 2.1.1: HCR schematic of detection and amplification stage
(Picture based on Choi et al, 2010). Detection Stage: Probe sets with initiator on the 5’ part
hybridize to mRNA target transcripts. Unbound probes are washed away. Amplification Stage:
Initiators are snapped cooled and added to the sample. As they consecutively anneal, then they
trigger a chain reaction and create a self-assembly of tethered fluorescent amplification polymers.
Excessive hairpins are immediately washed away from the sample. Experimental timeline, of an
example of three (3) different mRNA targets that are amplified simultaneously. Multiplex HCR
labeling is possible since different types of initiators trigger orthogonal HCR amplification
cascades of corresponding hairpins. Each set of initiators (System B2,B3,B4) has its
corresponding H1-H2 hairpin system that contains distinct fluorophores. Incubation times for
detection and amplification stage lasts 16hours each.
39
Two-stage multiplexed in situ hybridization using DNA HCR
Preparation Stage
1. Place 10-20 embryos into 1.5mL Eppendorf tubes. Remove as much egg water as possible
without touching the surface of the embryos.
2. Add 500uL of 4% PFA to each eppendorf tube and incubate at RT for 1hour or 4
o
C
overnight.
3. Wash samples 3 X 5 min with 1 mL of PBS to stop the fixation process. (Fixed embryos
can be stored as well at 4
o
C for further use)
4. Dehydrate and permeabilize embryos with a series of methanol (MeOH) washes (1 mL
volume for each eppendorf sample):
(a) 100% MeOH for 4 X 10 min
(b) 100% MeOH for 1 X 50 min
5. Rehydrate with a series of graded 1 mL MeOH/PBST washes for 5 min each:
(a) 75% MeOH / 25% PBST
(b) 50% MeOH / 50% PBST
(c) 25% MeOH / 75% PBST
(d) 5 X 100% PBST.
6. Store embryos at 4
o
C at this point, if need to stop the HCR protocol introduction.
Otherwise proceed in Detection stage
Detection stage
1. For each sample condition, move 10-20 embryos in 1.5 mL separate eppendorf tubes.
2. Pre-hybridize with 500 μL of probe hybridization buffer for 30 min at 37
o
C.
40
3. Prepare probe solution by adding 1 pmol of each probe (1 μL of 1 μM stock per probe) to
500 μL of probe hybridization buffer at 37
o
C.
4. Remove the pre-hybridization solution and add the probe solution.
5. Incubate the embryos overnight (12–16 h) at 37
o
C.
6. Remove excess probes by washing at 37
o
C with 500 μL of:
(a) 75% of probe wash buffer / 25% 5 X SSCT for 15 min
(b) 50% of probe wash buffer / 50% 5 X SSCT for 15 min
(c) 25% of probe wash buffer / 75% 5 X SSCT for 15 min
(d) 100% 5 X SSCT for 15 min
(e) 100% 5 X SSCT for 30 min.
Wash solutions should be pre-heated to 37
o
C before use.
Amplification stage
1. Pre-amplify embryos with 500 μL of amplification buffer for 30 min at room temperature.
2. Prepare 15 pmol of each fluorescently labeled hairpin by snap cooling in 5 μL 3uM hairpin
stock (already in 5X SSC buffer). Heat at 95
o
C for 90 seconds and cool to room
temperature on the benchtop for 30 min.
3. Prepare snap cooled hairpin solution by adding all snap-cooled hairpins (H1 and H2 for
each probe in your samples) to 500 μL of amplification buffer at room temperature.
4. Remove the pre-amplification solution and add the hairpin solution.
5. Incubate the embryos overnight (12–16 h) at room temperature.
6. Remove excess hairpins by washing with 500 μL of 5 X SSCT at room temperature:
(a) 2 X 5 min
41
(b) 2 X 30 min
(c) 1 X 5 min
7. Fix embryos with 4% PFA for 20minutes at room temperature, in order to stabilize the
hybridization of HCR probes and hairpins.
8. Wash three times with 500uL 1X PBS.
9. For optional counterstain with DAPI, add 1uL of 10mg/mL DAPI to 500uL 1X PBST
(0.1% tween-20). Place at 4
o
C overnight or 1hour in RT with gentle shaking.
10. Embryos can be mounted for imaging on 40mm thick Willco glass wells (HBST-5040) by
using low melt agarose in Danieu Solution.
42
Buffer recipes for short DNA probes HCR
Probe hybridization buffer For 40 mL of solution
30% formamide 12 mL formamide
5 X saline sodium citrate (SSC), 10 mL of 20 X SSC
9 mM citric acid (pH 6.0) 360 μL 1 M citric acid, pH 6.0
0.1% Tween 20 400 μL of 10% Tween 20
50 μg/mL heparin, 200 μL of 10 mg/mL heparin
1 X Denhardt’s solution 800 μL of 50 X Denhardt’s solution
10% dextran sulfate 8 mL of 50% dextran sulfate
Fill up to 40 mL with ultrapure H2O
Probe wash buffer Probe wash buffer
30% formamide 12 mL formamide
5 X saline sodium citrate (SSC), 10 mL of 20 X SSC
9 mM citric acid (pH 6.0) 360 μL 1 M citric acid, pH 6.0
0.1% Tween 20 400 μL of 10% Tween 20
50 μg/mL heparin, 200 μL of 10 mg/mL heparin
Fill up to 40 mL with ultrapure H2O
43
Amplification buffer
For 40 mL of solution
5 X saline sodium citrate (SSC) 10 mL of 20 X SSC
0.1% Tween 20 400 μL of 10% Tween 20
10% dextran sulfate 8 mL of 50% dextran sulfate
Fill up to 40 mL with ultrapure H2O
5 x SSCT For 40 mL of solution
5 X saline sodium citrate (SSC), 10 mL of 20 X SSC
0.1% Tween 20 400 μL of 10% Tween 20
Fill up to 40 mL with ultrapure H2O
50% dextran sulfate
For 40 mL of solution
50% dextran sulfate 20 g of dextran sulfate powder
Fill up to 40 mL with ultrapure H2O
Table 2.1.1: HCR solutions and buffer recipes compatible to short HCR DNA probe protocol
44
Probe
Number
DNA Probe Sequences for vegfr1 transcript
including Initiator for B2 Hairpin system
1 tgtgtatgaactcttgaagcaggaggaccgaaaaaagctcagtccatcctcgtaaatcctcatcaatcatc
2 cacggggacaacagtcaatctcggtagctaaaaaaagctcagtccatcctcgtaaatcctcatcaatcatc
3 ggatccaatactccagagaatcagttcaccaaaaaagctcagtccatcctcgtaaatcctcatcaatcatc
4 gtttggagatgaaggaatacctatggaggaaaaaaagctcagtccatcctcgtaaatcctcatcaatcatc
5 tctgtatctttagtcagaacacgtcctgaaaaaaagctcagtccatcctcgtaaatcctcatcaatcatcc
6 cagtaacatccaacactggagaactgaaccaaaaaagctcagtccatcctcgtaaatcctcatcaatcatc
7 gggtagagttttggtacgcctgatggcaaaaaaaaagctcagtccatcctcgtaaatcctcatcaatcatc
8 ctgcaatactggttacttttcttcccgcacaaaaaagctcagtccatcctcgtaaatcctcatcaatcatc
9 ggctatctgtgatgtagatgtagacagaagaaaaaagctcagtccatcctcgtaaatcctcatcaatcatc
10 gggttagtgactctgcatggaaagaccaagaaaaaagctcagtccatcctcgtaaatcctcatcaatcatc
11 tccggtggagaggaaacttaaccaacgagaaaaaaagctcagtccatcctcgtaaatcctcatcaatcatc
12 tgggactacgaataatgaatccctgcctgcaaaaaagctcagtccatcctcgtaaatcctcatcaatcatc
13 caacgatggtctcacaagaaaacagaccaaaaaaaagctcagtccatcctcgtaaatcctcatcaatcatc
14 actaaacccgtgctgttcaaatacacgtccaaaaaagctcagtccatcctcgtaaatcctcatcaatcatc
15 cttgatatgcgtttgctgataatggcggataaaaaagctcagtccatcctcgtaaatcctcatcaatcatc
45
16 gggattgtaaggacgctgtagaaaagcatgaaaaaagctcagtccatcctcgtaaatcctcatcaatcatc
17 agacaataactgtggtgtttgtctctcgttaaaaaagctcagtccatcctcgtaaatcctcatcaatcatc
18 gccatttctgtgcttgagtcgaatgaaaggaaaaaagctcagtccatcctcgtaaatcctcatcaatcatc
19 aggcggaaagatttctgtcctgcaaaagccaaaaaagctcagtccatcctcgtaaatcctcatcaatcatc
20 ccagatgatttcaggtgcagggaaagccttaaaaaagctcagtccatcctcgtaaatcctcatcaatcatc
21 agagaaaacccatccacatgataacgggagaaaaaagctcagtccatcctcgtaaatcctcatcaatcatc
22 ccagtaaggatagtatagattcctgcatccaaaaaagctcagtccatcctcgtaaatcctcatcaatcatc
23 accagtgtgatggtgaggttctggaaaagcaaaaaagctcagtccatcctcgtaaatcctcatcaatcatc
24 tggctgatagtcaagatgggattgtgggtgaaaaaagctcagtccatcctcgtaaatcctcatcaatcatc
25 ctgtcagaactccaacagtcttattcttccaaaaaagctcagtccatcctcgtaaatcctcatcaatcatc
26 atgtgacacagcgatagatgccagaaaccaaaaaaagctcagtccatcctcgtaaatcctcatcaatcatc
27 atagaaaggaatgtctagttcatctctgccaaaaaagctcagtccatcctcgtaaatcctcatcaatcatc
28 gctcttccactaaagatgcaacgaggccttaaaaaagctcagtccatcctcgtaaatcctcatcaatcatc
29 tgcctattggctatacatagcagacgaaggaaaaaagctcagtccatcctcgtaaatcctcatcaatcatc
30 ctgtgatttgactggttattctggaccatgaaaaaagctcagtccatcctcgtaaatcctcatcaatcatc
31 cagcccaaagtgcagagtgtgagagaactgaaaaaagctcagtccatcctcgtaaatcctcatcaatcatc
32 ctgctgtgtctagatgtatatgttctcctgaaaaaagctcagtccatcctcgtaaatcctcatcaatcatc
46
33 atgatcacttagatttccaagcagcaccggaaaaaagctcagtccatcctcgtaaatcctcatcaatcatc
34 agggcagtggagagtgatagagttgcttacaaaaaagctcagtccatcctcgtaaatcctcatcaatcatc
35 ttcttataccatgttatgtgtggctgtggcaaaaaagctcagtccatcctcgtaaatcctcatcaatcatc
36 gtaattcggtcaatgtgaagagtcccttccaaaaaagctcagtccatcctcgtaaatcctcatcaatcatc
37 ttggtggcttgacaggtataaaatccctggaaaaaagctcagtccatcctcgtaaatcctcatcaatcatc
38 gggattcagacgaattttgaacccaaatgtaaaaaagctcagtccatcctcgtaaatcctcatcaatcatc
39 gtttggatgtttgagtttccgaatcagcagaaaaaagctcagtccatcctcgtaaatcctcatcaatcatc
40 tgcaagatgatcggcaggtactctgctttgaaaaaagctcagtccatcctcgtaaatcctcatcaatcatc
Table 2.1.2: DNA HCR probe sequences against vegfr1 transcript
47
Probe
Number
DNA Probe Sequences for vegfr2 transcript
Not including Initiator for B3 Hairpin system
1 gcggtcgcacctgccctcgggagttgtggataaaaaaagtctaatccgtccctgcctctatatctccactc
2 tcaccgaagaaccatctctatcatatcaactaaaaaaagtctaatccgtccctgcctctatatctccactc
3 tcctcttaaaacctcagtcaaagccttctttaaaaaaagtctaatccgtccctgcctctatatctccactc
4 acactcatttgcagaggatcaactgcactgtaaaaaaagtctaatccgtccctgcctctatatctccactc
5 tgcagtcacaagaagtccagcgatcatgaataaaaaaagtctaatccgtccctgcctctatatctccactc
6 cagatgatgttaaaagaggggtacagtgggtaaaaaaagtctaatccgtccctgcctctatatctccactc
7 ctacttaaaaggctaacaatggaaattaaataaaaaaagtctaatccgtccctgcctctatatctccactc
8 gagtttcataaggagcggatcaatcgtccttaaaaaaagtctaatccgtccctgcctctatatctccactc
9 acatacacgtgcacaggatcaattgaattttaaaaaaagtctaatccgtccctgcctctatatctccactc
10 gctgttcctgcgcccgacagagtcttgtggtaaaaaaagtctaatccgtccctgcctctatatctccactc
11 ctatcgctctgtcaaaccaggagaggggtctaaaaaaagtctaatccgtccctgcctctatatctccactc
12 ttggtattcccatgccgaacattacctggttaaaaaaagtctaatccgtccctgcctctatatctccactc
13 gataaaggatgtaaacagcaaggttggatttaaaaaaagtctaatccgtccctgcctctatatctccactc
14 gaacttggcaaccggaccatgagaatcccttaaaaaaagtctaatccgtccctgcctctatatctccactc
15 agaggcacaagatacctctacgataggctgtaaaaaaagtctaatccgtccctgcctctatatctccactc
48
16 gtttccagaaaccatacactcggctatgagtaaaaaaagtctaatccgtccctgcctctatatctccactc
17 tgcagaatctgaccaatcaggatgtaaacataaaaaaagtctaatccgtccctgcctctatatctccactc
18 taaaagatgatggcactctaattattgaaataaaaaaagtctaatccgtccctgcctctatatctccactc
19 agcagcagcaacattcctgtggattatgcttaaaaaaagtctaatccgtccctgcctctatatctccactc
20 gtgtgaccgtctaccatatgacagcaacaataaaaaaagtctaatccgtccctgcctctatatctccactc
21 caagatttcaacatgcaaaacagtggctgttaaaaaaagtctaatccgtccctgcctctatatctccactc
22 ctgctaggagcctgcacaaagcgtggcggctaaaaaaagtctaatccgtccctgcctctatatctccactc
23 caagtctcaggacggtaaggctgtgcgttctaaaaaaagtctaatccgtccctgcctctatatctccactc
24 ctccggcttcattgaagataagagctactgtaaaaaaagtctaatccgtccctgcctctatatctccactc
25 gtatccaccgtgatctggctgcacgtaacataaaaaaagtctaatccgtccctgcctctatatctccactc
26 tgtccgcaaaggagacgctagacttccctttaaaaaaagtctaatccgtccctgcctctatatctccactc
27 ggactgctggcatggagaaccatctcagagtaaaaaaagtctaatccgtccctgcctctatatctccactc
28 caaagcagacccctcaaaccagagtcccactaaaaaaagtctaatccgtccctgcctctatatctccactc
29 aacaagatccacgagggtgggcagtcagactaaaaaaagtctaatccgtccctgcctctatatctccactc
30 cagttctgctggagggcgaaatggacaaattaaaaaaagtctaatccgtccctgcctctatatctccactc
31 acaccacccgtctgacatgctctgcgctggtaaaaaaagtctaatccgtccctgcctctatatctccactc
32 acatttcctgtacacttctgtcaagttagttaaaaaaagtctaatccgtccctgcctctatatctccactc
49
33 ttacactaacagcactgtttccccattgaataaaaaaagtctaatccgtccctgcctctatatctccactc
34 ttgctaaacgtattctttcagagctcgctataaaaaaagtctaatccgtccctgcctctatatctccactc
35 ttgatcatcagcatacttatgatgcacatttaaaaaaagtctaatccgtccctgcctctatatctccactc
36 ttagttaaaattcagtttaagatcttcaactaaaaaaagtctaatccgtccctgcctctatatctccactc
37 acacaacagttacactgcatcgtgtacttttaaaaaaagtctaatccgtccctgcctctatatctccactc
38 tgctactaaagcttgcagggcaggctatgataaaaaaagtctaatccgtccctgcctctatatctccactc
39 gagattatcgtattggttaagccagattattaaaaaaagtctaatccgtccctgcctctatatctccactc
40 N/A
Table 2.1.3: DNA HCR probe sequences against vegfr2 transcript
50
Probe
Number
DNA Probe Sequences for vegfr3 transcript
Not including Initiator for B4 Hairpin system
1 gggttttagatattctagcaaacctgcgcgattttcacatttacagacctcaacctacctccaactctcac
2 cgtaaaatctctcttcatttccaggtttccattttcacatttacagacctcaacctacctccaactctcac
3 cctgagaagaagggaatcccaatccaaatcattttcacatttacagacctcaacctacctccaactctcac
4 gtcaagggtgggtggactcatagaaaacccattttcacatttacagacctcaacctacctccaactctcac
5 agtgtgtcgttagcgttaatcacaagctggattttcacatttacagacctcaacctacctccaactctcac
6 aaactccactttactcaaagactcttcaggattttcacatttacagacctcaacctacctccaactctcac
7 ctccttcagcctgatttctctataccctggattttcacatttacagacctcaacctacctccaactctcac
8 ggcctgggcattggtaagtattaagatcttattttcacatttacagacctcaacctacctccaactctcac
9 cagctttaatgtccttgtagaagcagcggtattttcacatttacagacctcaacctacctccaactctcac
10 gctctgggtctcgaacaaacacaaaaatgcattttcacatttacagacctcaacctacctccaactctcac
11 gagtctgttatgaagatggtctccatgtcgattttcacatttacagacctcaacctacctccaactctcac
12 ggtcaggatctgaaaccagacatggtacttattttcacatttacagacctcaacctacctccaactctcac
13 gctctggatacggcactaacgagaagagagattttcacatttacagacctcaacctacctccaactctcac
14 ccctttttattattccaggtgaccacactgattttcacatttacagacctcaacctacctccaactctcac
15 ggtggaagtgttctgaatgatatgcctgggattttcacatttacagacctcaacctacctccaactctcac
51
16 ggctgttttggactgagatggagcagtagaattttcacatttacagacctcaacctacctccaactctcac
17 gcccaatgacctggacaacatagattgatgattttcacatttacagacctcaacctacctccaactctcac
18 ggcgagtcttcaggaaacagcttgaactcaattttcacatttacagacctcaacctacctccaactctcac
19 cactggaaatccacaccagtgttgaagtcgattttcacatttacagacctcaacctacctccaactctcac
20 gacgttacgcagaggctgtaaactcgccaaattttcacatttacagacctcaacctacctccaactctcac
21 gtgaatgtttctgattgagaggatactggaattttcacatttacagacctcaacctacctccaactctcac
22 gctctcgtttcatttccagagtattagcccattttcacatttacagacctcaacctacctccaactctcac
23 cggacagatttttgtccctcttttgcctctattttcacatttacagacctcaacctacctccaactctcac
24 ggaccttaaacctgcttgaattcttgctgcattttcacatttacagacctcaacctacctccaactctcac
25 agggctatttttcaggaccagcatgtattcattttcacatttacagacctcaacctacctccaactctcac
26 gaggtggaacgttgactatgagagtaaagtattttcacatttacagacctcaacctacctccaactctcac
27 catccagttgtgacagaaaggactctggtcattttcacatttacagacctcaacctacctccaactctcac
28 ccaccatctgagtcaatgtgtcaatactctattttcacatttacagacctcaacctacctccaactctcac
29 ctggcacactggcattctgaataaccacacattttcacatttacagacctcaacctacctccaactctcac
30 gggctccatctctatatcgaatccttcaggattttcacatttacagacctcaacctacctccaactctcac
31 cgcaggttctcataggtgaaattatctgcatattttcacatttacagacctcaacctacctccaactctcac
32 gttgctggtggtctgaaaagatagttgtccattttcacatttacagacctcaacctacctccaactctcac
52
33 tcctgtagctgaatgttggtaatgttaagcattttcacatttacagacctcaacctacctccaactctcac
34 tgggatgtatttgcggtgacaatgtttcacattttcacatttacagacctcaacctacctccaactctcac
35 ctcgctcacatttaccgtatgattagttggattttcacatttacagacctcaacctacctccaactctcac
36 gggttggttatctttaaaccaggacagctgattttcacatttacagacctcaacctacctccaactctcac
37 tacattggttttgtcatcagagccaatcacattttcacatttacagacctcaacctacctccaactctcac
38 acccagaaaaagatggcgatgactcctgtgattttcacatttacagacctcaacctacctccaactctcac
39 cactcgcttgacattgcagaagatgacaagattttcacatttacagacctcaacctacctccaactctcac
40 gtccatgatgatggacagatagcctgtcttattttcacatttacagacctcaacctacctccaactctcac
Table 2.1.4: DNA HCR probe sequences against vegfr3 transcript
53
2.2 Confocal Microscopy and Imaging Conditions
All samples were mounted in agarose molds and immobilized with 1% LM agarose,
facilitating stage mounting and lateral imaging conditions. Zebrafish samples were imaged with
Zeiss 780 Upright confocal microscope (Zeiss,Inc.) and 40x LD C-Apochromat NA 1.1 W Corr
objective. During classical confocal acquisitions all samples have been acquired with build-in
basic selection of MBS, secondary dichroics, emission filters and detectors, optimized for each
fluorophore emission spectra. Pinhole is set at 1 AU and z- stack space at 1μm or less, depending
on occasional oversampling conditions for a subset of samples (Figure 2.2.1 A-B).
In the case of hyperspectral acquisition, a set of 32 channels with 8.9nm spectral space has
been utilized during each experimental acquisition (Figure 2.2.1 C). It is highly important to
conserve the same filter set configurations, when imaging hyperspectrally, so that pure single
fluorophore control spectra can simultaneously be used in downstream Hybrid Unmixing software,
presented in section 2.3 of this Chapter. For semiquantitative purposes presented in the following
chapters a hyperspectral acquisition has been performed that allows full spectral collection from
405nm-780nm, according to Table 2.2.1.
Finally, multiple images are simultaneously imaged as tiles and overlapping by 10%-pixel size on
x-y direction. By using Imaris Stitcher, Bitplane, any of 32 channels of hyperspectral data can be
used to perform alignment on x-y axis due to stage drifting or on z-due to hysteresis. Specific
puncta size conditions can be used to fit the Gauss analysis provided in the software. Furthermore,
HYSP post-processed image can be loaded to Imaris Stitcher, for a secondary validation of tile
acquisition and alignment procedure. (Figure 2.2.2 A-B).
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Figure 2.2.1 Confocal Microscopy apparatus
(A) Close look on confocal microscopy experimental apparatus where the objective is placed on
the top of a confocal microscope (Zeiss 780) and whole mounted embryos are imaged when
mounted with LM agarose. (B) Embryos are imaged on the left lateral plane with special focus on
the vessels from ISV 15 down to the end of their body tail, as shown in red dashed box. (C)
Hyperspectral acquisition of all available 32 channels with Zen Software and subsequent tile
stitching with Imaris Stitcher, before or after Hybrid Unmixing. Tiles must overlap by 10% to
avoid possible stage drifting and result a successful experimental alignment.
55
Figure 2.2.2 Imaris stitcher on tile images
(A) Hyperspectral Tile Image aligned with Imaris Stitcher, Bitplane. Any of 32 channels can be
used to perform alignment on x-y axis due to stage drifting or on z-due to hysteresis. Specific
puncta size conditions can be used to fit the Gauss analysis provided in the software. Red dashed
circle indicates cross section of four tile images, (B) HYSP post-processed image can be loaded to
Imaris Stitcher, Bitplane for a secondary optional “check point” of the alignment procedure.
Yellow dash line indicates cross section of four tile images. (A-B) White lines indicate 10% image
acquisition overlap and endothelial vessel types are labeled in blue as ISV,DA and PCV.
56
Table 2.2.1: Basic Filter Sets: confocal acquisition on Zeiss 780 Upright
Laser Excitation Color Basic Filter Sets
405nm DAPI MBS 458/514/594 & -405/+760
488nm eGFP MBS 488/561/633 & -405/+760
Alexa 514 * MBS 458/514/594 & -405/+760
514nm eGFP * MBS 488/561/633 & -405/+760
Alexa 514 MBS 458/514/594 & -405/+760
594nm Alexa 594 MBS 458/514/594 & -405/+760
Alexa 647 * MBS 488/561/633 & -405/+760
633nm Alexa 594 * MBS 458/514/594 & -405/+760
Alexa 647 MBS 488/561/633 & -405/+760
57
2.3 Background on Hybrid Unmixing, HYSP
Hyperspectral unmixing and HYSP software is a direct result of scientific work directed
from Dr. Francesco Cultrale and his students in the Fraser Group, during the years that I conducted
my PhD work. It was recently developed as a commercially developed scientific tool (Cutrale et
al., 2017) and it continuously adapts to become a widely applicable user-friendly software that
allows efficient signal unmixing, precise identification of AF signal from tissue autofluorescence
in various wavelengths and background removal. In this section I am presenting the biological
applications of this software, as it was used for efficient signal unmixing of my experimental
samples containing DAPI, eGFP, Alexa 514, Alexa 594, and Alexa 647. (Figure 2.3.1-2). It was
highly important to combine the capacities of HYSP signal unmixing to establish a pipeline of
reproducible sample signal, removal of background and establishing a semi-quantification pipeline
that could utilize all multiplex capacities available by in situ HCR staining (Figure 2.4.1).
By coherently identifying the resulting fluorophore signals, we were able to truly focus on
cluster creating corresponding to actual transcript signal and establish a semi-quantification
pipeline. Challenging fluorophore spectra, as presented in Figure 2.3.1, were able to unmix and
identify separately. This advanced hybrid inmixing imaging software has the capacity to further
expand multiplexing and semi-quantification imaging processing by allowing pure fluorophore
signal to be utilized without introduction of noise bias.
58
59
Figure 2.3.1 HYSP unmixing for eGFP and Alexa 514 overlapping spectra
Characteristic example of two emission spectra that are partially overlapping and are not
commonly used simultaneously in normal confocal acquisitions with basic filter sets, but their
separation is straightforward with the use of hyperspectral acquisition and HYSP software signal
unmixing. (A) Excitation (dashed) and emission (continuous) spectra is collected hyperspectrally
in whole mount zebrafish embryos stained with Alexa 514 labelled HCR probes (vegfr1) and eGFP
transgenic expression in all endothelial vessels. (B) Hyperspectral phasor plotting the 2
nd
harmonic
of their corresponding Fourier coefficients, S for the real component, G for the imaginary
component of the Fourier Plane. Red dots represent the spectral selection exhibited in next panel.
(C) Emission spectra for eGFP (blue), Alexa 514 (yellow) and AF/Background (red). (D) Separate
channels after signal unmixing with HYSP software for eGFP, Alexa 514 and AF/Background
signal
60
61
Figure 2.3.2 Control signal for hyperspectral unmixing
Hyperspectral Phasors for eGFP, Alexa 514, 594 and 647 when excited with 488nm, 514nm,
594nm and 647nm, respectively (left column). Hyperspectral signatures are presented in S and G
Fourier space coordinates, when selecting the 2
nd
harmonic of the represented signal. Selected
regions on the phasors are represented as emission spectra (middle column). Background and
autofluorescence is pseudo colored in red in each emission spectra graph, while blue graphs
indicate eGFP, Alexa 514, Alexa 594 and Alexa 647 emission spectra. Raw hyperspectral
acquisition images are presented in grey scale (right column) and serve as guide sample pictures
and signature verification.
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2.4 Data Analysis Pipeline and Methods of Image Rendering (Imaris, Bitplane)
Methods workflow for establishing semi-quantification
In following chapter 3, a biological interpretation of experimental semi-quantification
analysis is performed. All data have been processed with HYSP software and tile aligned when
applicable with Imaris Stitcher. In particular, all fluorophores utilized in analysis presented in
Chapter 3, were unmixed with control single spectra, as presented in Figure 2.3.2, using a recently
developed hybrid unmixing adaptation on HYSP software (upcoming paper, Chang et al., 2021).
This novel adaptation allowed further expansion of semi-quantification image analysis presented
in Chapter 3.
Finally, resulting spectra were processed with image rendering software, Imaris, Bitplane
to create district rendering volumes corresponding to various vessel types and transcript cluster
groups (Figure 2.4.3). Characteristic examples of iso-volume creation are presented in Figure
2.4.3 and guide the reader into the required steps of iso-volume formation and rendering). Each
resulting vessel type was co-processed with total number of transcript clusters (Figure 2.4.4)
resulting a set of vessel specific cluster volume statistics that could be extracted and further
processed with custom Matlab scripts. A characteristically pilot plot is presented in Figure 2.4.5
and explains the difference between cluster populations in terms of mean intensity and size.
Methods workflow for establishing semi-quantification
Step 1: Establish the HCR configuration system for multiple labeling of vegfr transcript types
according to Part 2.1, Chapter 2. Use commercial (Molecular Instruments, Inc) hairpin systems
B2,B3,B4 that allow orthogonal amplification.
63
Step 2: Hyperspectrally acquire all available spectra of 32-channels using Zeiss 780 Upright
confocal microscope. Each fluorophore is excited with separate laser lines according to Part 2.2,
Chapter 2 section
Step 3: Use HYSP signal unmixing software (Cutrale et al., 2017) by using pure fluorophore
spectra as shown in Figure 2.3.2 and proceed with Phasors represented in Figure 2.4.2 of this
section.
Step 4: Use Imaris Stitcher when working with multiple tile images composing a larger whole
mounted embryo sample and double check if clusters are properly aligning. Use secondary signals
from each phasor to troubleshoot possible misaligning issues
Step 5: Use Imaris volume rendering to create total vessel volumes, identify subsets of ISV, DA,
PCV and their subsequent transcript as presented in Figure 2.4.3
Step 6: Use Imaris volume rendering again to create transcript cluster volumes, identify subsets
of ISV, DA, PCV clusters as presented in Figure 2.4.4. For their subsequent active nuclear sites
and fingerprint nuclear volumes of single and double transcript expression follow steps presented
in workflow of Figure 2.4.5.
Step 7: Export all statistical analysis data and iso-volume information with custom made Matlab
scripts that read resulting Imaris files. Proceed with necessary semi-quantification analysis for
cluster volume vs mean intensity plots, tissue specific and global CVO definition and finally t-test
score analysis to define significant differences among samples, as in Section 2.5, Chapter 2
64
65
Figure 2.4.1: Methods workflow for semi-quantification of transcript clusters
In situ HCR labelling: Probes against vegfr1, vegfr2,vegfr3 are designed, compatible with HCR
systems B2-Alexa 514, B3-Alexa 594, B4-Alexa 647 respectively (Molecular Instruments, Inc).
Hyperspectral Acquisition: Whole-mount embryos are hyperspectrally imaged by collecting 32-
channels for 5x2 tiled frames (Zeiss 780, Upright). . HYSP Unmixing: DAPI, eGFP, Alexa 514,
Alexa 594, Alexa 647 control spectra for signal unmixing (HYSP software). Safety Check: Tile
frame alignment (Imaris Stitcher); correction of possible hysteresis effects if present. Imaris
volume rendering: Manual iso-volume separation of DA vessels; subsequent identification of ISV,
PCV vessels (Imaris). Transcript Cluster Creation: Automatic cluster creation for each vegfr
transcript (Imaris). Data Extraction: Custom Matlab scripts for iso-volume statistical data
extraction and transcript cluster statistical analysis.
66
67
Figure 2.4.2: Hyperspectral Unmixing of multiplex HCR samples
Characteristic example of five (5) different fluorophores in a multiplex HCR-stained whole mount
zebrafish sample. By using clear spectra from previously acquired single labeled samples, HYSP
software has the capacity to purely unmix their respective signal from AF or Background.
Secondary signal is collected as well and used when appropriate to verify cluster overall and
possible stage hysteresis during acquisition
68
69
Figure 2.4.3: Imaris workflow for creating iso-volumes
Schematic guide example walking the user through necessary Imaris iso-volume rendering for
identification of ISV, DA, PCV and their subsequent transcript cluster volume for species A and
B in each subsequent vessel type. (A) Open all available channels, (B) Selecting channel of
interest. eGFP signal in particular for this example, (C) Create an iso-surface on Imaris, which
marks the boundaries of eGFP signal on vessels, (D) Mask all channels of interest, respectively
(E) Carefully enclose punctuated patterns and clusters of all species on interest (magenta and cyan
here). (F) Mask again all channels of interest within EC cluster volume, so that species puncta can
be contoured (G) Manually contour the DA so that it can be separated from ISV and/or PCV, as
separate quantification volumes (H) Repeat step E, but for cluster species A-B in DA (I) ) Repeat
step F, but for cluster species A-B in DA (J) Select the ISV iso-volume created as a residue of step
G (K) Carefully enclose iso-volume clusters of all species as in step E or H, (L) Mask all channels
of interest indicating cluster volume withing ISVs. Scale bar 20μm
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Figure 2.4.4: Transcript cluster creation
Transgenic eGFP signal expressing in all ECs is used to create vessel iso-volumes. DA arterial
trunk has been manually dissected with Imaris Tool Selection and separated from ISV and PCV
vessel boundaries. The result is the identification of all three (3) vessel type volumes for
downstream analysis. In this example, we create the subcellular transcript clusters of vegfr1 that
exhibit more distinct texture (granulated), while vegfr2 exhibit less distinct texture (more unified).
Statistical data are exported with the use of customed Matlab script and plotted as mean intensity
of clusters vs transcript cluster volume. Grey dots represent the different classifications of vegfr1
and vegfr2 clusters as expected to be represented in this sample plot
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Figure 2.4.5: Sample Cluster Quantification Schematic
Iso-volume information statistic generated by Imaris rendering can be extracted plotted in terms
of mean intensity [a.u] versus cluster volume [μm3] size. Clusters that are classified as “dim” range
in terms of mean intensity from 1-10, as “moderate’ from 10-100 and as “bright” from 100-1000
on a logarithmic scale. Clusters that exhibit more granulated pattern will appear on the left side of
the graph, such as those of vegfr1, while vegfr2 that have a more unified expression will appear on
the right side of the graph. Representation varying on the x axis exhibits a larger thus more unified
cluster volume.
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Figure 2.4.6: Identifying nuclear active transcripts and nuclear volume fingerprint
Iso Probes against vegfr1, vegfr2,vegfr3 are designed, compatible with HCR systems B2-Alexa
514, B3-Alexa 594, B4-Alexa 647 respectively and used for orthogonal transcript amplification.
Step 1: iso-volumes of important vessel types, mask all eGFP vessel types, create ISV DA PCV
and identify all cluster iso-volumes for each transcript type. Step 2: Identifying active nuclear dots,
mask all nuclei by using DAPI signal, assume minimum x,y size of 1μm and apply model of PSF-
elongation on z-axis at 2 μm depth. Background subtraction for defining cluster edges. Mask for
overlapping clusters with nuclei volumes. Step 3: Creating nuclear volume active transcript
fingerprint. Identify various subsets of nuclei volumes enclosing single positive and double
positive signals. Color-code the resulting fingerprint volumes.
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2.5 Mathematical Background of Graphical Representations
Cluster Volume Occupancy
Our focus is to perform semi-quantification in subcellular transcript patterns and transcript
cluster formation within samples, while examining their reproducibility. For this purpose, we
introduced the concept of Cluster Volume Occupancy that represents density measurement of
transcript clusters in endothelial vessels. More specifically the CVO is defined as tissue specific,
when referring to each vessel region in particular (point density). On the other hand, the global
tissue CVO quantifies a generalized cluster measurement referring to total vessel volume (global
density). The above definitions are presented in specialized equations referring to ISV, DA,PCV
vessels and total volume of all vessel types (VT) in Table 2.5.1 on this section.
Cluster Volume Occupancy example of vegfr1 clusters in DA vessels
The total volume taking part on this example of CVO calculation corresponds to the total
DA vessel volume VDA (green) as presented in Figure 2.5.1, while other vessels do not contribute
to this example (marked as grey). Transcript cluster iso-volumes of vegfr1 are defined as yellow
(panel ii-iii). Each cluster has an individual subsequent iso-volume (V1, V2, V3, V4 etc.) within its
DA population. All individual volumes are summed in order to further calculate CVOs
DA
, as shown
in Table 3.5.1 for i=vegfr1 and DA vessels. Finally, DA tissue specific CVO of vegfr1 transcript
is defined as the ratio between the sum of all its vegfr1 clusters divided by the total DA volume
(green).
Performing t-test scores
For semi-quantification of significant changes among various embryos presented in
distribution plots of Chapter 3, we introduced the definition of unpaired or paired t-test scores with
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95% confidence, we would accept as non-significant changes the samples pairs that score 𝑝 >
0.05 (white box), while the ones that fail the null hypothesis (grey box) and exhibit statistically
significant changes have 𝑝 ≤ 0.05. Single asterisk p* refers to values 0.01 < 𝑝 ∗≤ 0.05 and
represents significant changes, while double asterisk 𝑝 ∗∗≤ 0.01 to even higher significant
changes. Statistical analysis and plotting were performed with custom Matlab Scripts
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Figure 2.5.1: Definition of Cluster Volume Occupancy of vegfr1 in Dorsal Aorta (DA).
(A) Different endothelial volumes indicating the boundaries between ISVs, DA and PCV. The
total volume taking part on this example of CVO calculation corresponds to the total DA vessel
volume VDA (green) while other vessels do not contribute to this example and so are marked as
grey. (B) Transcript cluster iso-volumes of vegfr1 (yellow). Each cluster has an individual
subsequent iso-volume (V1, V2, V3, V4 etc.) within its DA population. All individual volumes are
summed in order to further calculate CVOs
DA
. (C) DA specific CVO of vegfr1 transcript is defined
as the ratio between the sum of all its clusters divided by the total DA volume (green). Scale bar
at 15 μm.
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Table 2.5.1: List of equation defining tissue specific & global Cluster Volume Occupancy
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CHAPTER 3 Exploring the role of vegfr1, vegfr2 and vegfr3
transcripts during angiogenesis and vasculogenesis
3.1 Introduction
In this chapter, we focus on vegfr transcript localization and active nuclear transcript
expression during first and second wave of spouting angiogenesis [26-34hpf] in various
endothelial tissues during vascular development. Two morphogenetic processes are taking place
simultaneously and influence the formation of vertebrate blood vessels; vasculogenesis and
angiogenesis (Isogai et al., 2001). [Chapter 1, Section 1.1]
In order to better understand vegfr transcript profiles on various endothelial tissues we need
to consider the axes of different morphogenic processes that are taking place throughout the
embryo’s vascular development. At each developmental stage of the embryo, along the
dorsal/ventral and anterior/posterior axis vasculogenesis is taking place simultaneously. However,
the angiogenic sprouting of arterial or venous derived intersomitic vessels during first and second
wave of angiogenesis respectively, is following the zebrafish’s somite developmental gradient
from anterior to posterior (Holley et al., 2006) [Chapter 1, Section 1.2]. In existing literature, this
somite developmental gradient is referred as segmentation clock; a form of genetic oscillator
[References here]. The first six (6) anterior somites form every 20min, while the 24 posterior
somites form every 30min (Schubert et al, 2001). In a similar pattern, intersomitic vessels are
sprouting during first and second wave of angiogenesis, following the developmental flux of
genetic oscillator from anterior to posterior.
Studying the vegfr transcript localization dynamics at a specific developmental stage of the
zebrafish embryo requires consideration of the sequential angiogenic sprouting (anatomical time)
from anterior to posterior as well as specific morphogenic effects along various stages of zebrafish
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embryo development (developmental time). In order to understand the specific subcellular patterns
or vegfr transcript “fingerprint” of the endothelial vessels, we can focus on the same vessel among
stages or move from posterior (newer vessels) to anterior (older vessels) at a specific stage [Figure
3.1.1]. We focus on transcript expression of three (3) district types of vegfr receptor family; vegfr1,
vegfr2 and vegfr3. As described in Chapter 1, this dynamical interplay between those three
receptors is crucial in order to understand the underlying mechanisms that drive angiogenesis,
vasculogenesis and lymphangiogenesis at later stages.
Instead of solely focusing to the protein level of those receptors, we are choosing to follow
their mRNA sequence in order to fully capture the lifetime and spatial subcellular localization of
those important nucleotide sequences. We are interested to capture not only when they are actively
transcribed but as well their transcript clustering and distribution, within the endothelial vessels.
Thus, we are focusing on the main important stages of mRNA dynamics, which are the following:
generation of nascent transcript in the nucleus, which is produced once the corresponding receptor
gene is actively transcribed. Once the single stranded mRNA is correctly processed then it
concludes its export towards the cytoplasm in the form of ribonucleoprotein complexes (mRNPs).
There we follow the subcellular clustering of those transcript and their preferential expression in
the developing vasculature.
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Figure 3.1.1: Defining the developmental wave of ISVs within the same stage or along various
stages during vascular development. (A) Lateral embryo representation, marking example of
anterior and posterior vessels presented below. (B & D) Anatomical time defines the
morphological frame under which ISV develop under the same stage, which includes newer
sprouting vessels (white gradient) on the posterior part towards the tail and older vessels (black
gradient) towards the anterior part above the yolk extension (AYE). (C & E) Developmental time
exhibits a flux that follows the traditional morphological development of ISV during
vascularization of the developing embryo.
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3.2 Multiplex HCR labelling of vegfr transcripts in the developing vasculature
The first wave of angiogenic sprouting is taking place around 24hpf [Reference here],
arising from dorsal aorta (DA) derived endothelial cells. This primary angiogenic network is
emerging bilaterally from the DA at each vertical somite boundary (Isogai et al., 2003). With the
use of Tg(kdrl:eGFP) transgenic line, we can follow the eGFP expression withing the developing
vasculature and examine the angiogenic blood vessels network formation. The eGFP fluorescent
protein is expressed under the vegfr2 receptor ortholog gene promoter, referred as kdr-like, kdrl,
(Quinn et al., 1993). It recapitulates its corresponding receptor expression along endothelial cells
during zebrafish embryo development. Figure 3.2.1, panel A is presenting a lateral view of
Tg(kdrl:eGFP) zebrafish embryo at 26hpf. The eGFP labelled endothelial vessels are imaged with
confocal microscopy, post-imaging processed and combined with a widefield screening
microscopy image of an embryo at the same stage. This panel presents important anatomical
features of the embryo, as well as important eGFP labelled features of the vascular network.
Intersomitic vessels (ISV) are counted from anterior to posterior and labelled with grey arrows,
showing the 1
st
ISV,10
th
ISV, 20
th
ISV (upper part), the already lumenized DA & forming PCV
(lower part). At 26hpf, the dorsal aorta (DA) is fully formed and endothelial cells in posterior
cardinal vein (PCV) are forming the sub intestinal plexus. Strike box (red) highlight region of
vasculature is highlighting the 16
th
and 17
th
ISV above the yolk extension, later shown in panel B.
Important vegfr transcripts are labelled simultaneously with multiplex in situ hybridization
chain reaction (HCR) in whole mount zebrafish embryos at 26hpf (for further background info on
HCR see Part 1.6, Chapter 1, for probe design; Part 1.1, Chapter 2) corresponding to the first
wave of angiogenesis. In Figure 3.2.1, panel B(i-iv) is exhibiting a distinct subcellular localization
pattern of vegr1, vegfr2 and vegfr3 transcripts. All different vegfr transcripts reveal endothelial
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tissue specificity and various expression levels among different endothelial tissues of the
developing vasculature. By observing these important and distinct transcripts of the vascular
endothelial growth factor receptor family, we conclude in separate quantitative observations.
Vegfr2 transcript expresses in all endothelial cells, independent of their vascular anatomy. Vegfr1
transcript is selectively expressing in artery derived endothelial cells and more specifically in the
already lumenized dorsa aorta (DA). It is visible as well throughout the developing intersomitic
vessels (ISVs). On the contrary, vegfr3 transcript is specialized in venous derived endothelial
tissues, which correspond to the post cardinal vein (PCV) plexus undergoing vascularization.
It is present as well towards the middle part (stalk cells) of the developing intersomitic
vessels (ISVs).In Figure 3.2.1, panel C, we attempt to quantify the qualitative observations
concluding from multiplex HCR labelling and confocal imaging, presented in panel B. On these
semi-logarithmic dot plots, the x-axis represents transcript cluster volume [=μm
3
], while the y-axis
shows the mean fluorescence intensity values (12-bit) of each cluster in arbitrary units [a.u.]. In
order to appropriately generate them, we spectrally unmixed its fluorescent signatures, created
surface contouring of their subcellular cluster boundaries and extracted intensity data with
conserved spatial information with the use of Imaris, Bitplane (for HYSP and Imaris rendering;
see Chapter 2). Post processing and graphical representation, we defined three (3) distinct regions
of classification; dim (0-1au), moderate (1-100au) and bright (100-1000). All clusters that arise as
large aggregates with bright signal appear as a single unified cluster count above the red line, while
slightly visible ones are positioned between blue-red line. Average cluster volume should be
considered as well, when estimating the average cluster intensity of each vegfr transcript on each
endothelial region.
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Quantitative results from panel C are in direct correlation with HCR data presented in part
B of this figure and we were able to expand our conclusions. Vegfr-1 is expressing moderate to
high in ISV and very strongly in DA, while its expression becomes significantly lower in PCV.
Vegfr-2 is highly expressed in ISV, DA and slightly moderate to high in PCV. Vegfr-3 is highly
expressed in PCV and stalk cells of ISV, while its signal turns dimer into DA. At this point,
observing and quantifying one single embryo at 26hpf, is not enough to draw several general
conclusions on endothelial tissues and their preferential transcript localization.
In order to appropriately quantify the expression levels presented in Figure 3.2.1, panel A-
C and uniformly characterize these results in various embryos at this stage, we introduced the
definition of global or specific Cluster Volume Occupancy (CVO), extensively presented in
Chapter 2 and Materials and methods of this chapter. In Figure 3.2.2 panel i-iii, we present the
characteristic example of vegfr1 clusters and how we define the tissue specific CVO when this
transcript is expressed in dorsal aorta (DA) . As described in Chapter 2, we use hybrid unmixing
(HYSP) in order to spectrally unmix the various fluorescent signals as well as occasional sample
autofluorescence or background signal. We further create iso-volumes in order to create
compartments contouring the ISVs, DA and PCV respectively. In Tg(kdrl:eGFP) embryos all
endothelial tissues are strongly expressing eGFP. Anatomical boundaries between different vessel
types can be distinguished with data 3D representation with Imaris, Bitplane and can be manually
dissected into subsequent volumes. Further features on Imaris, allow selective isolation of cluster
volumes within defined endothelial surface boundaries, allowing a wide variety of data generation
and extraction.
By using custom build algorithms (Matlab) we have the capacity to extract (from Imaris
platform) a collection of variables referring to vegfr1, vegfr2 and vegfr3 transcript clusters and
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their spatial resolution within endothelial tissues. Tissue specific CVOs is calculated in order to
exhibit the volumetric cluster ratio specific to the adduced vessels (ISV, DA or PCV) among N=7
(see equations in Table 2.5.1 of Materials and Methods). Global CVOg is calculated in order to
exhibit a non-tissue normalized version of cluster volume occupancy that refers to the total
endothelial surface volume (ISV,DA and PCV combined). This specific cluster volume occupancy
intrinsically includes the size variation of adducted vessels and describes cluster distribution as it
is presented in Figure 3.2.1
Definition of significant changes presented in distribution plots of Figure 3.2.3, we
introduced the definition of unpaired or paired t-test scores (for further definitions, see Materials
and Methods of this chapter). With 95% confidence, we would accept as non-significant changes
the samples pairs that score 𝑝 > 0.05 (white box), while the ones that fail the null hypothesis
(grey box) and exhibit statistically significant changes have 𝑝 ≤ 0.05. Single asterisk p* refers to
values 0.01 < 𝑝 ∗≤ 0.05 and represents significant changes, while double asterisk 𝑝 ∗∗≤ 0.01 to
even higher significant changes.
In conclusion, Figure 3.2.3 presents the general trends of vegfr transcript expression at
26hpf. In ISVs both vegfr2 & vegfr1 are expressed with a highly clustered pattern, but vegfr3 is
only expressed on the stalk cells. There is high variation of vegfr1 cluster formation along different
samples in ISVs. Further, DA exhibits strong clustering of vegr2 & vegfr1 while vegfr3 clusters
are almost absent. PCV exhibits less localization of vegfr2 & vegfr1, but vegfr3 has a higher more
granulated population of clusters DA and PCV are the vessel where most subcellular transcript
cluster changes are occurring
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85
Figure 3.2.1: vegfr transcripts exhibit granulated and distinct localization pattern in various
vessels during angiogenesis and vasculogenesis
(A) Widefield lateral view of Tg(kdrl:eGFP) zebrafish embryo at 26hpf combined with confocal
acquisition of eGFP labelled developing vasculature. Strike box (red) highlights the region of
vasculature around the adjunct area below the 16
th
and 17
th
ISV. Scale bar at 150 μm. (B) Multiplex
fluorescence in situ HCR of vegr1, vegfr2 and vegfr3 transcripts in Tg(kdrl:eGFP) embryos
presented in composite (i) and single channel (ii-iv) panels respectively. Composite panel (i)
includes merged image of vegfr1 transcript (yellow), vegfr2 (magenta), vegfr3 (cyan), eGFP label
of all vessels (EC, green) and DAPI staining of nuclei (blue). Stroke white line marks the
boundaries between DA and PCV. Scale bar at 15 μm. (C) Scatter plots of vegfr transcript clusters
within the three types of vasculature, ISV, DA and PCV shown in (B).
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87
Figure 3.2.2: Cluster Volume Occupancy of vegfr transcripts at 26hpf, for multiple embryos
(A) Tissue specific CVOs is calculated in order to exhibit the volumetric cluster ratio specific to
the adduced vessels (ISV, DA or PCV) among N=7. (B) Global specific CVOg is calculated in
order to exhibit the volumetric cluster ratio which is referring to the total volume of endothelial
vessels (ISV, DA and PCV combined)
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Table 3.2.1: t-test scores for tissue specific sCVO
Performing unpaired t-test on the CVOS various transcript types (vegfr1, vegfr2 or vegfr3) between
vessel types ISV, DA, PCV in N=5 embryos. Single star (*) refers to 0.01 0.05 (white box), while the ones that fail the null hypothesis (grey box) and exhibit
statistically significant changes have 𝑝 ≤ 0.05. Single asterisk p* refers to values 0.01 < 𝑝 ∗≤
0.05 and represents significant changes, while double asterisk 𝑝 ∗∗≤ 0.01 to even higher
significant changes.
Performing t-test on the CVOG of same vessel types (ISV, DA, PCV or total vessels VT)
among different combinations of transcript (vegfr1/2, vegfr2/3, vegfr1/3). With 95% confidence,
we would accept as non-significant changes the samples pairs that score 𝑝 > 0.05 (white box),
while the ones that fail the null hypothesis (grey box) and exhibit statistically significant changes
have 0.01 < 𝑝 ∗≤ 0.05. On this table, vegfr2/3 pair exhibits its most significant change in global
CVO within ISV and DA. Tissue specific Cluster Volume Occupancy (CVOs) is defined for each
vessel type in equations 1-3. The sum of each population of clusters in ISV, DA or PCV is divided
by its subsequent vessel volume for each vegfr1, vegfr2 or vegfr3 transcript separately. Equations
1-3 are marked in panel B.
In ISV vessels, vegfr1 transcript expression is significantly lower in dnm1l mutants, vegfr3
is becoming almost zero and vegfr2 is remaining the same. In DA, vegfr1 transcript in dnm1l is
becoming lower, while vegfr3 is closer to zero as before. In PCV, their trend reverses with vegfr1
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tending towards zero sCVO value and vegfr3 getting lower than before. In both DA and PCV,
vegfr2 does not exhibit significant change between WT and dnm1l mutants. Tissue specific CVOS
is calculated in order to exhibit the volumetric cluster ratio specific to the adduced vessels (ISV,
DA or PCV) among N=4 samples for dnm1l mutants. In dnm1l mutants, vegfr1 has its highest
value in ISV, decreasing in DA and reaching almost zero at PCV. Vegfr2 maintains similar CVOS
values between ISV and DA but is becoming lower in PCV. Vegfr3 is almost zero in ISV and DA
and exhibit its highest expression in PCV.
Maximum intensity projection image of DA and PCV past the end of yolk extension, which
is placed bellow the anterior trunk vessels at ISV 17. Transcripts as detected by in situ HCR
pseudo-colored: vegfr1(yellow) and vegfr2 (magenta). Vegfr1 transcript expression during ISV
development on the upper panel, while joined expression of vegfr2 and vegfr1 is presented on the
lower panel. Maximum intensity projection image of DA and PCV past the end of yolk extension,
which is placed bellow the anterior trunk vessels at ISV 17. Transcripts as detected by in situ HCR
pseudo-colored: vegfr3(cyan) and vegfr2 (magenta). Vegfr3 transcript expression during ISV
development on the upper panel, while joined expression of vegfr2 and vegfr1 is presented on the
lower panel.
Global Cluster Volume Occupancy (CVOs) is defined for each vessel type in equations 4-
7. The sum of each population of clusters in ISV, DA or PCV is divided by the total volume of all
vessel types (VTV, where VTV= VISV+ VDA+ VPCV) volume for each vegfr1, vegfr2 or vegfr3
transcript separately. Equations 4-7 are marked in panel C. In ISV vessels, vegfr1 transcript
expression is significantly lower in dnm1l mutants, vegfr3 is becoming almost zero and vegfr2 is
remaining the same. In DA, vegfr1 transcript in dnm1l is becoming lower, while vegfr3 is closer
to zero as before. In PCV, their trend reverses with vegfr1 tending towards zero sCVO value and
122
vegfr3 getting lower than before. In both DA and PCV, vegfr2 does not exhibit significant change
between WT and dnm1l mutants.
Tissue specific CVOS is calculated in order to exhibit the volumetric cluster ratio specific
to the adduced vessels (ISV, DA or PCV) among N=4 samples for dnm1l mutants. In dnm1l
mutants, vegfr1 has its highest value in ISV, decreasing in DA and reaching almost zero at PCV.
Vegfr2 maintains similar CVOS values between ISV and DA but is becoming lower in PCV.
Vegfr3 is almost zero in ISV and DA and exhibit its highest expression in PCV.
Maximum intensity projection image of DA and PCV past the end of yolk extension, which
is placed bellow the anterior trunk vessels at ISV 17. Transcripts as detected by in situ HCR
pseudo-colored: vegfr1(yellow) and vegfr2 (magenta). Vegfr1 transcript expression during ISV
development on the upper panel, while joined expression of vegfr2 and vegfr1 is presented on the
lower panel. Maximum intensity projection image of DA and PCV past the end of yolk extension,
which is placed bellow the anterior trunk vessels at ISV 17. Transcripts as detected by in situ HCR
pseudo-colored: vegfr3(cyan) and vegfr2 (magenta). Vegfr3 transcript expression during ISV
development on the upper panel, while joined expression of vegfr2 and vegfr1 is presented on the
lower panel.
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CHAPTER 4 Future Directions
4.1 On studying VEGFR receptor dynamics and vegfr transcript subcellular
signatures in other model organisms
Although most human pathologies and diseases have mostly been studied widely in
mammal systems such as rodents, lower vertebrates such as zebrafish (Danio rerio) have gained
extreme popularity and attention as a model animal system. Most scientists have long utilized
animal models to mimic or study basic processes in much simple animal organisms, narrowing
down the complexity of cellular pathways signals, organ function and gene expression (Goldsmith
et al., 2012). Most scientific studies have been done in rats and mice that belong to the higher
mammalian systems closer to humans. Those model systems are established as the most essential
research tools for clinical trials, drug screen and understanding human pathologies. However,
important discoveries have been made using invertebrate systems such as fruit flies (Drosophila
Melanogaster) and C.elegans (Caenorhabditis elegans). Drosophila was the model organism that
contributed to findings such as Toll-like receptors (TLRs) and its special linkage to the nuclear
factor kappa-light-chain enhancer of activated B cells (NF-κB) (Hansson et al., 2005). C.elegans
are the innovative model organism and genetic system that helped the scientific community shade
light into RNA interreference; a widely used technique under which double stranded RNA is
introduced and results targeted gene silencing (Fire et al., 1998). Oppositely to drosophila and
C.elegans, zebrafish is a vertebrate organism with higher physiological, molecular and
morphological characteristics closer to a mammalian system, but with the ease of maintenance and
use of a lower organism (Goldsmith et al., 2012).
In this PhD thesis we used the vertebrate system of zebrafish (Danio rerio) as an excellent
higher model organism system for studying its endothelial vascular development (Lawson et al,
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2002). It is small, has fast development, low cost for growing, low opacity in embryos and its
genome is fully mapped. Zebrafish homology with human genome, including non-coding regions
is 60 per gene totally across all genes (Kardash, 2012; Goldsmith et al., 2012). In Chapter 1, we
presented further general info about the key regulators of vascular development and sprouting
angiogenesis. In this part we would like to link those findings into other model organisms such as
mice, which have been extensively used to elucidate the role of VEGFR receptors and its ligands.
Interestingly, the drosophila system has been reported to have a VEGFR receptor and VEGF
ligand homologue expressed in developing and mature hemocytes but not endothelial cells (Heino
et al, 2001). But this invertebrate system, although it is a common model organism for
developmental processes and biomedical science in general, is lacking the traditional arrangement
of vascular system. Instead of a branching endothelial network or arteries and veins, it has a
tracheal system for oxygen diffusion. Tracheal arrangement is highly branched as well with a
similar mechanism to vascular branching morphogenesis. It is governed by FGF signaling pathway
which is analogous to the traditional VEGFR signaling system in vertebrates (Forbes et al.,1997).
Thus, extensive search on literature identifies mostly mice and zebrafish as the most ideal
candidates to study vascular development and sprouting angiogenesis of endothelial cells, while
VEGFR expression in drosophila is limited.
The key regulators of vascular development and sprouting angiogenesis in vertebrates
(referred to Chapter 1) are the vascular endothelial growth factor receptors (VEGFRs) and their
ligands. All endothelial cells and a subgroup of haematopoietic cells, express VEGFR1 (Flt1),
VEGFR2 (Flk1/KDR) and VEGFR3 (Flt4) (Ferrara et al., 1999, Neufeld et al., 1999). They belong
to the general family of tyrosine kinase receptors, which are activated by a ligand induced binding,
subsequent dimerization and autophosphorylation. The extracellular part of VEGFRs is consisted
125
by seven (7) immunoglobulin (Ig) homology domains and as a structure display specificity for a
given group of VEGF ligands that cause hetero- or homodimerization of VEGFR receptors
(Eriksson et al., 1999). VEGFR2 receptor VEGF-A ligand are major components of endothelial
cells from their angioblasts (mesodermal precursors), which migrate in order to form de novo
vascular cords through vasculogenesis, as well as vessels and capillaries through sprouting
angiogenesis; endothelial vessel formation from pre-existing ones (Risau, 1997) (see Part 1.1-1.3
in Chapter 1). Important fact to underline is that angioblasts are common embryonic precursors of
both hematopoietic and endothelial cells (Choi et al., 1998, Ziegler et al., 1999) expressing
VEGFR2, which explains why in invertebrates such as Drosophila, the VEGFR/VEGF
homologues are expressed in developing and mature hemocytes (Heino et al, 2001).
Apart from the zebrafish model that we extensively covered in this present Thesis, the
scientific field has traditionally studied VEGFR receptor functionality, their adjacent signal
pathways and important drug screening/development in mouse models. Genetic mutant studies in
mice have underlined the crucial role of VEGFR/VEGF signaling pathway in vascularization,
angiogenesis, blood formation and lymphangiogenesis. Homozygous mutant embryos in the main
receptor driving vascular development, VEGFR2 or its primary ligand VEGF-A immediately
results in early embryonic lethality at E8-9 stage (Ferrara et al., 2004; Thurston et al.,2008), while
partial deletion of the VEGF-A allele results in lethality at E11-12. Those gene knockout
experiments in mouse models have shown that VEGFR2 is essential not only for endothelial cell
differentiation and early vascular development, but further haematopoietic cell function in the
embryo. Angioblasts lacking VEGFR2 are unable to reach the correct embryo location to pre-form
blood islands structures and thereby are unable to respond to the appropriate haematopoietic
signals (Shalaby et al., 1997).
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Furthermore, in mice VEGFR3 and VEGFR1 receptors have been studied and found
essential as well for vascular and lymphatic development, but not as crucial as VEGFR2.
Depending on partial or total allele silencing, possible lethality phenotypes are observed in later
mouse embryonic stages. Deletion of VEGFR3 receptor or potential blockage of its corresponding
VEGF-C ligand, results in dysfunctional development of the lymphatic system (Thurston et
al.,2008). Homozygous mice lacking VEGF-C allele do not develop lymphatic vessels and
lethality appears at E15.5-17.5 (Kärkkäinen et al., 2004), while partial allele loss results in
lymphedema. Furthermore, VEGFR3 receptor mutant mice embryos exhibit a severe blood
formation and vascular phenotype, way before the lymphatic system is developed. Major defects
are apparent in remodeling of the primary vascular plexus (Dumont et al., 1998) and major defects
in hematopoiesis (Hamada et al., 2000). VEGFR3 double mutant embryos die early in
development as they fail to develop a cardiovascular network and successful hematopoiesis;
phenotypes that are leading towards the conclusion that VEGFR3 receptor is essential in the early
stages on endothelial cell differentiation and blood cell formation (Kärkkäinen et al., 2000).
Moreover, VEGFR1 receptor is interconnected with VEGFR2 receptor expression and
holds an important role in endothelial cell proliferation, successful migration during early
embryogenesis and embryonic survival. In early scientific literature, it was shown that VEGFR1
binds to VEGF-A with a 10-fold higher affinity than VEGFR2 homodimers achieve in CMT3 cells
(Terman et al., 1992). VEGFR1 forms heterodimer complexes with VEGFR2 that have stronger
signaling capacity than VEGFR1 or VEGFR2 homodimers alone (Huang et al., 2001).
Importantly, VEGFR1 homodimer transmits weak signals of mitogenesis in endothelial cells
(Ferrara et al., 2003) due to low autophosphorylation upon ligand binding. Mice homozygous
127
knockouts of VEGFR1 die during embryonic development at E8.5 due to elevated production of
angioblasts and disorganized, over sprouting vascular development (Fong et al., 1995).
In addition, VEGFR1 is important during the early embryonic stages were angioblasts
migrate and differentiate, but not as crucial as VEGFR2. It also acts as a sink for the abundance of
VEGFR-A, working synergistically with VEGFR2 receptor in order to balance the correct ratio of
stalk and tip cells during sprouting, proliferation and elongation of artery derived ECs. These
findings are verified as it has been showed that expressing an incomplete truncated version of
VEGFR1, which lacks the intracellular TRK domain, results in mice embryos that develop
normally (Hiratsuka et al., 1998) but might develop moderate phenotype of impaired angiogenic
sprouting.
In mouse models, vegfr1, vegfr2 and vegfr3 transcript expressions have been studied with
traditional chromogenic in situ transcript hybridization techniques, limited to the cases where
specimen section or sample opacity was allowing spatial resolution and efficient probe
hybridization to the mRNA transcript targets. In mice, further transcript quantification studies have
been limited to the transcript levels acquired from qPCR; a technique that requires invasive
specimen removal and lacks spatial resolution. The most prominent tissue that has been
extensively studied due to its low opacity, in terms of detailed VEGFR receptor expression, is the
mouse retina (Stahl et al, 2010). Conventional sectioning techniques for tissues are two-
dimensional and limit the extensive study of anatomical structures compared to intact tissues
(Richardson et al., 2015). Extracting spatial information from an intact biological specimen with
intrinsic high opacity has been a fundamental challenge in developmental biology and
biomedicine; light cannot penetrate deeper tissue layers, which restricts imagined depth, resolution
and contrast.
128
Recent development in tissue CLARITY techniques (Chung et al, 2013) transform tissues
of interest into a hydrogel embedded specimen that preserve proteins or nucleic acids to their
original place while have the capacity to completely remove lipid layers. CLARITY techniques
lower specimen opacity by achieving optical transparency (Tomer et al., 2014) and increase
antibody diffusion during immunostaining (Nehrhoff et al., 2016). CLARITY and similar
techniques have successfully been used in most mouse organs (Lee et al., 2014) and allow several
rounds of labelling, washing or probe hybridization steps as proteins and nucleic acids are strongly
bound to the hydrogel matrix that samples are embedded.
Finally, combining CLARITY with powerful multiplex in situ hybridization techniques
such as HCR (Choi et al., 2010) , with orthogonal transcript labelling and high signal to
background ratios, we can achieve efficient in situ hybridization transcript labelling in whole
mouse organs. Similar experiments have not been reported so far and exciting research results can
be created in combination with cutting edge optical microscopy. With combination of CLARITY,
HCR, immunohistochemistry, advance optical microscopy and specialized 3D rendering tools,
new paths of studying VEGFR receptors and their corresponding vegfr transcripts can be created.
Paths that will elucidate vasculogenesis, spouting angiogenesis and corresponding pathologies in
a combined level of protein and nucleic acid expression with spatial resolution in intact tissues for
the first time.
129
130
Figure 4.1.1 Graphical representation of VEGFR receptors in three different levels of central
dogma of biology
The genetic information encoding vegfr1,vegfr2 and vegfr3 genes exists on the cell nucleus and
has been studied on DNA level in various higher vertebrates. The receptor proteins encoding those
genes, VEGFR1, VEGFR2,VEGFR3 are presented on the cell membrane of activated cells and
has been extensively studied throughout bibliography on a protein level. Little is known on the
mRNA level of those transcripts and existing literature is limited in traditional chromogenic
techniques that are lacking spatial resolution and in situ labelling sensitivity in the case of low
transcript expression. This PhD thesis is elucidating the subcellular transcript localization and
cluster formation of those important vegfr transcripts.
131
4.2 Membraneless organelles, P-bodies, Stress granules and others;
potential association with vegfr transcript regulation
Nascent transcripts (mRNAs) bind and actively associate to a collection of RNA-binding
proteins (RBPs) and microRNAs. They collectively create a dynamic machinery that determine
their subcellular fates. The life circle of an mRNA includes the following components: expression
of nascent transcript as the gene is actively transcribed inside the nucleus; exported from the
nucleus in the form of messenger ribonucleoprotein particles (mRNPs); transportation of mRNPs
into subcellular regions for translation into protein, storage and degradation of mRNA transcripts.
The most important parts of those mRNA transcripts that mediate their subcellular interaction with
the cell machinery and association with proteins, are found on the cis elements of the 5’- or 3’-
untranslated transcript regions (UTRs).
These regulatory elements, which are named RNA binding proteins (RBPs), are either
unique to a single RNA transcript species or are found in a subset of RNA molecules coding
proteins that belong the same family with functional similarities. Individual RBPs have a broad
spectrum of subcellular functions; influence mRNA metabolism, transcript decay/stabilization,
targeted subcellular localization and translation rate (Anderson et al., 2015). The availability and
functionality of those RBPs of mRNPs complexes that are assembled around mRNA transcripts in
a co-operative manner, determine mRNA stability, transcript localization and targeted translation
in order to allow a dynamic control over protein synthesis.
With the use of advance confocal microscopy and signal reconstruction techniques we were
able to identify anatomical subcellular characteristics and patterns of mRNPs. Those mRNA
transcript particles form discrete subcellular granule like patterns that can provide precise control
over targeted protein synthesis and operation inside the cell. More than three decades now, it has
132
been becoming clear that subcellular targeting of mRNAs is essential for many cellular functions
(Holt et sl., 2009). There are several main reasons why localizing an mRNA at a particular cellular
site could be advantageous; (a) Energy conservation by subcellular localization of transcripts, since
one single mRNA molecule can produce multiple rounds of protein expression, (b) preventing
displacement of proteins during conditions that favor their translocation, (c) facilitating the local
expression of proteins and their molecular complexes by exhibiting high local mRNA
concentration, (d) allocate the control of gene expression by permitting specific subcellular local
control of translation in response to extracellular stimuli in a timely fashion.
Despite their anatomical punctuated subcellular pattern, RNP granules are unique in their
molecular composition, formation and function. Some of those have been classified as Cajal
bodies, nucleoli, germ granules, stress granules, P-bodies, GW-bodies and their formation depends
on various systems of RNA-protein complexes, membraneless organelles and liquid-liquid phase
separation (LLPS) puncta (Lin et al., 2015). From this wide pool of RNP granules we can mainly
divide them into two distinct categories: nuclear RNPs and cytoplasmic. There is a wide RNA
binding protein collection that regulates mRNA stability, degradation, targeted localization and
translation in the cytoplasm. All these cellular mechanisms are spatially regulated by the formation
of mRNP containing cytoplasmic granules that influence cell metabolism and function.
Perturbations on the system that regulates mRNA granule formations can lead to pathological
conditions observed in neurological, immune related or infectious diseases (Anderson et al., 2015).
There are three distinct groups of cytoplasmic RNP granules; germ granules, stress
granules and P-bodies (Kulkarni et al., 2017). Germ granules are cytoplasmic non-membrane
bound clusters of proteins and RNAs, unique for germ cell development (Wang et al., 2014). Stress
granules are RNA-protein aggregations that appear when a cell is under a stress response. Their
133
proposed function is to protect untranslated mRNAs from extreme stress conditions either for
further proper storage or by facilitating re-initiation of translation (Wheeler et al., 2016).
134
4.3 Current methods that allow selective mutation on important parts of vegfr
transcripts for expanding current PhD results
Traditional functional experiments and selective transcript silencing in zebrafish was
taking place with the use of morpholinos, complementary partial sequences (nucleotide analogs)
that recognize transcript parts of interest that inhibit translation or proper mRNA splicing
(Summerton & Weller, 1997) . Current gene editing technologies such as CRISPR-Cas9 can act
more specifically to the gene level and introduce specific mutations in zebrafish genome,
transforming the era of reverse genetics for this animal model (Lieu et al, 2019).
In this current PHD thesis, we focus on the mRNA level of the three most important vegfr
transcripts and how their expression associates with disrupted endocytic components such as
dynamin-1. Furthermore, we followed the anatomical time of cluster expression between Anterior
and Posterior parts of ISV vessels undergoing sprouting angiogenesis from the DA. Developmental
time of posterior vessels during angiogenesis and vasculogenesis reveals important info with
spatial resolution during 1
st
and 2
nd
wave of angiogenesis.
Finally, we established a methods workflow combining existing commercial software and
techniques (HCR, HYSP,Imaris,Matlab) in order to establish a semi-quantification baseline for
cluster volume occupancy and pattern expression. With the use of CRISPR-Cas9 systems, this
PhD can be expanded further into precise characterization of vegfr transcript role in cytoplasmic
transcript expression and mRNA regulation
135
4.4 Translational approaches: From studying model organisms to new anti-
angiogenic drug development
Angiogenesis is a normal physiological process during embryonic development, growth
and wound healing. It is controlled by a series of angiogenesis-stimulating growth factors and
inhibitors. When the balance between those factors is disturbed, the result is abnormal vessel
growth that leads to many pathological conditions. Inadequate vessel formation that is mainly
caused by over-expression of angiogenesis inhibitors or inadequate production of angiogenic
growth factors on a metabolic active tissue may result in inhibition of repair mechanisms or other
critical functions. Several diseases or conditions, such as coronary artery disease, ischemic stroke
and chronic wounds are the result of insufficient angiogenesis. On the other hand, excessive
vascular growth contributes to numerous disorders such as inflammatory diseases, retinopathy,
age-related macular degeneration, rheumatoid arthritis and cancer metastasis. As we have already
mentioned, VEGF-induced neovascularization also plays a major role in pathological processes
such as tumor growth and metastasis (Folkman, 1995).
136
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Abstract (if available)
Abstract
An increased understanding of the molecular mechanisms that controls angiogenesis and vasculogenesis is critical for the development of novel therapeutic strategies against vascular diseases. These diseases can re-activate embryonic vascular formation signaling events. An ideal animal model to study vascular development is zebrafish (Danio rerio) due to its transparency and easy in vitro/in vivo experimental manipulation. During embryogenesis, blood vessel precursors undergo a series of complex morphogenesis events and elaborate in vascularization of adult zebrafish body. A key regulator of vascular development is VEGF receptor 2 (VEGFR2), which plays a crucial role in all aspects of normal, and pathological vascular endothelial cell dynamics. Its dynamic expression interconnects with other vascular endothelial growth factor receptors, such as VEGFR1 & VEGFR3. This PhD thesis aims to elucidate the dynamics of vegfr transcript localization during angiogenesis of ISVs and vasculogenesis of DA and PCV; how their cytoplasmic transcript localization can interconnect with receptor endocytosis and patterns of transcript subcellular localization during 1st and 2nd wave of angiogenesis. With the use of confocal microscopy, cutting edge multiplexed in situ hybridization chain reaction (HCR), hyperspectral signal un-mixing (HYSP) and post imaging analysis, a coherent mapping of mRNA expression is performed within intact whole mount zebrafish embryos. Furthermore, multiplex labelling of vegfr1, vegfr2 and vegfr3 is allowing a thorough transcript localization analysis in developing vasculature between 1st and 2nd wave of angiogenesis.
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Asset Metadata
Creator
Dimotsantou, Maria Eleni
(author)
Core Title
Exploring the role of vegfr transcripts during vascular development in the zebrafish embryo
School
Viterbi School of Engineering
Degree
Doctor of Philosophy
Degree Program
Biomedical Engineering
Degree Conferral Date
2021-05
Publication Date
05/10/2023
Defense Date
01/15/2021
Publisher
University of Southern California
(original),
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Tag
anatomical time,angiogenesis,artery derived endothelial cells,cluster volume occupancy,confocal microscopy,DA,developmental time,first wave of angiogenesis,hyperspectral imaging,HYSP,image rendering,imaging,imaging analysis,ISV,morphological development,mRNA,OAI-PMH Harvest,PCV,second wave of angiogenesis,semi-quantification analysis,transcript localization,vascular development,vasculogenesis,vegfr1,VEGFR-1,vegfr2,VEGFR-2,vegfr3,VEGFR-3,vein derived endothelial cells,vessel formation,zebrafish
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Fraser, Scott (
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), Finley, Stacey (
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), McCain, Megan (
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), Phillips, Carolyn (
committee member
), Trinh, Le (
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)
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dimotsan@usc.edu,mdimotsa@gmail.com
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Tags
anatomical time
angiogenesis
artery derived endothelial cells
cluster volume occupancy
confocal microscopy
DA
developmental time
first wave of angiogenesis
hyperspectral imaging
HYSP
image rendering
imaging
imaging analysis
ISV
morphological development
mRNA
PCV
second wave of angiogenesis
semi-quantification analysis
transcript localization
vascular development
vasculogenesis
vegfr1
VEGFR-1
vegfr2
VEGFR-2
vegfr3
VEGFR-3
vein derived endothelial cells
vessel formation
zebrafish