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Development of the lymphatics in the eye

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Development of the lymphatics in the eye

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DEVELOPMENT OF THE LYMPHATICS IN
THE EYE

By

Yifan Wu

A Thesis Presented to the
Faculty of the USC Graduate School
University of Southern California
In Partial Fulfillment of the
Master of Science
Biochemistry and Molecular Biology

August 2019

Copyright 2019

2
Acknowledgements
I would like to express my deepest appreciation to everyone who has supported my research and coursework
throughout my two years in pursuit of a Master’s degree. First, I would like to give my deepest gratitude to my
parents, who have supported me emotionally and financially for past of my life, especially in the years I’ve been
abroad, and for guiding me to become an honest and hardworking person.
I want to give my utmost appreciation to my principal investigator, Dr. Young-Kwon Hong, and all my lab
members. Without Dr. Hong’s support and guidance, I would not have been able to start my project, find a new
direction from the failures and finish the project successfully. He is such an inspiring, creative and helpful professor
who has guided me into the scientific field and whom I will undoubtedly continue to learn from in the future. I
would like to acknowledge Eunson Jung, who knows everything about research and life, and has helped me out
whenever I meet trouble. I also want to express my appreciation to Dr. Dongwon Choi, the most dedicated
scientist, who generously pardoned my faults and taught me so much, to Eunkyung Park, who has the most skillful
hands in the world and patiently taught me the experimental techniques step by step, to Dr. Sunju Lee, who
organized everything in our lab and allowed me to work efficiently on my project, to Sophia Zhao and Shrimika
Madhavan, who accompanied and helped me in work and in life, to Young Jin Seong, who showed me every
technique he knew about the eyeballs. In addition, I also want to thank to the numerous mice I used, who
contributed their lives to make good figures for my project.
Last but not least, I would like to express my appreciation to my committee members, Dr. Young-Kwon Hong,
Dr. Ching-Ling (Ellen) Lien, Dr. Sandy Zhang-Nunes, and Dr. Wange Lu, for sparing their value time to attend my
committee meetings and give me guidance about my project, thesis and my future career.
3

Table of Contents
Acknowledgements ………………………………………………………………………………………………………………………....... 2
Table of Contents …………………………………………………………………………………………………………………………........ 3
List of Figures ………………………………………………………………………………………………………………………………......... 4
Abstract ………………………………………………………………………………………………………………………………………......... 5
1. Introduction ………………………………………………………………………………………………………………………………..... 6
2. Materials and Methods …………………………………………………………………………………………………………….... 15
3. Results ……………………………………………………………………………………………………………............................... 18
4. Discussion ………………………………………………………………………………………………………............................... 25
5. Reference ………………………………………………………………………………………………………............................... 28

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List of Figures
Figure 1. Structure of the human eyeball and anatomy of the limbus ................................................ 7
Figure 2. Comparison between human and murine eyeballs .............................................................. 8
Figure 3. Lymphatic vessels development .......................................................................................... 11
Figure 4. Blood vessel distribution in the eye ..................................................................................... 13
Figure 5. Schematic of human sample ................................................................................................ 15
Figure 6. Eye clover processing .......................................................................................................... 16
Figure 7. Conjunctival and limbal lymphatics origin from one route at the nasal side ....................... 18
Figure 8. Postnatal development of the conjunctival and limbal lymphatics ..................................... 20
Figure 9. Differentiation between limbal and conjunctival lymphatics .............................................. 21
Figure 10. Different development patterns of lymphatics and blood vessels in the eye ................... 22
Figure 11. Human limbus has polarized distribution of lymphatics .................................................... 24

5

Abstract
The limbus is a transitional zone between the transparent cornea and the opaque sclera, and serves as
the surgical incision site for diseases such as cataracts and glaucoma. Current anatomical knowledge
indicates that the cornea is devoid of any vasculature while the limbal area is filled with lymphatic vessels,
which are connected to the conjunctival lymphatic network. Under inflammatory conditions, the pre-
existing lymphatic vessels extend to the cornea, facilitating the exit of antigenic material from the cornea
to the lymph nodes to induce an adaptive immune response. It has been shown in adult mice that the
nasal side of the limbus bears a predominant pattern of lymphatic distribution; however, the origin of
limbal lymphatics and their development is not clear. This project focuses on these questions and
extends the exploration into how limbal blood vessels and lymphatic vessels differ from each other in
origination and development using eyeballs collected from mouse and rat pups. After extensive imaging,
it was found that the lymphatic vessels in the eye has one single origination from the nasal side, and
develop bidirectionally to enclose the cornea postnatally, while blood vessels are from multiple routes
and are well developed before birth.

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1. Introduction
1.1 Eye Anatomy
1.1.1 Human eye anatomy
The eye is one of the most complex and sensitive organs in the human body. It is divided into two major
chambers: (a) the anterior chamber: the chamber between the cornea and lens, which is filled with
aqueous humor and (b) the vitreous chamber: the chamber between the lens and sclera, which is filled
with vitreous humor (1) (Figure 1A). The cornea serves the dual function of providing physical protection
for the interior of the eye and causing around two thirds of the light refraction needed for vision (2).
Between the cornea and the sclera, there is a transitional zone called the limbus (Figure 1B). Structurally,
the limbus demarcates the transparent cornea and the opaque sclera and connects the corneal epithelia
externally to the conjunctival epithelia (Figure 1 C&D). Functionally, the limbus is the often surgical
incision site into the anterior chamber for treating cataract and glaucoma; it is also where the aqueous
humor outflows to keep the peripheral corneal nourished (3). On the upper layer of the cornea, the
conjunctiva connects the front of cornea and the interior of the eyelid and provides lubrication and
protection for both the outer surface of the eye and inner surface of the eyelid (4) (Figure 1C).

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Figure 1. Structure of the human eyeball and anatomy of the limbus. 1A. Structure of the human eyeball.
The human eyeball is constructed with two chambers: the anterior chamber between cornea and lens, and
the vitreous chamber between lens and sclera. 1B. Front view of the eye, showing the position of the
limbus. 1C. Side view of the eye, showing the positions of the limbus and conjunctiva. 1D. Anatomy of the
limbus. The limbus is usually considered to be the area between the two red lines, which is between the
cornea and sclera. Figure 1D is modified from Van Buskirk, 1989 (3).
8

1.1.2 Murine eye anatomy
The mouse eyeball is similar in structure to the human eyeball, although its relative dimensions are
different. Human eyeballs are mainly filled with vitreous, while the murine eyeballs are predominantly
occupied by the lens (Figure 2.)(5). Compared to humans, mice have thicker lens and shorter focal length
(the distance between the middle of lens and retina), which results in brighter but smaller images in the
eye (6). Despite these differences in proportion, the murine eye is structurally analogous enough to the
human eye that it still serves as a good model for studying the development, maintenance and wound
healing, etc. of the cornea. Thus, many types of transgenic mouse models have been developed for
human eye diseases related research, each specific to focuses such as the infections, morphogenesis and
ocular oxidative pressure (7).

1.2 Lymphatic vessels: Function and Development

Figure 2. Comparison between
human and murine eyeballs.
Human and murine eyeballs bear
similar structure but different
dimensions. For example, the
murine eyeballs have bigger lens
and shorter focal length. However,
mice eyeballs are still good models
for many human eyeball studies,
such as development and diseases
related researches. Figure 2 is
modified from Skeie et al., 2011 (5).
9
The lymphatic system is a critical vascular system in human body that exists in most organs, with
exceptions including the cornea, epidermis, cartilage and central neural system. It has three main roles:
cooperating with blood vessel system to maintain the fluid homeostasis, immune response, and fat
metabolism. The fluid in the semipermeable blood vessels extravasates to the interstitial space under
the hydrostatic pressure produced by the heart. Most of this part of fluid will be reabsorbed by initial
lymphatics, and the unique primary valves between lymphatic endothelial cells ensure the reabsorbed
fluid cannot flow back (8, 9). Dysfunction of lymphatics leads to the accumulation of fluid in tissues and
results in edema. The fluid, large proteins, immune cells and lipids are able to enter into the lymphatic
vessels against the pressure gradient, and are transported unidirectionally through lymphatic vessels
due to the existence of luminal valves (8). The lymph is then transported to collecting lymphatic vessels
and is returned to blood vessels; thus the circulation between blood and lymphatic vascular systems is
accomplished and fluid homeostasis is maintained (10). Though the lymphatic system is not considered
a part of the immune system, it is crucial for immune response. It primarily serves as trafficking system
for the antigen and immune cells such as leukocytes and activated antigen-presenting cells. It has been
reported that lymphatic endothelial cells in lymph nodes secrete cytokines as well (11). As part of lipid
metabolism, lymphatic vessels provide active lipid transport from the small intestine to the circulation
in the form of lacteals in the intestinal villi. Lymphatic dysfunction under this functional aspect can lead
to systematic consequences in lipid metabolism (12).

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Lymphatic development starts at around embryonic day 9 (E9) in mice (13) and at around weeks 6 to 7
in human (14). The development is a stepwise process, involving competence, specification,
determination, remodeling and maintenance (Figure 3.)(15). The initiation of lymphatic endothelial cells
(LECs) specification is from Prox1 expression in a subpopulation of cardinal vein endothelial cells (ECs),
which requires transcription factors Sox18 and COUP-TFII to make the vein ECs to be competent in
response to Prox1. Functional inactivation of any one of these genes leads to loss of LECs differentiation
and lymphatic vasculature cannot form (13, 16, 17). The Prox1 expressing LEC progenitor cells then bud
from the cardinal veins and arrive at surrounding mesenchyme to form primitive lymph sacs (18). During
the budding process, there are both cell and nuclear shape changes (19), and cells begin to express
additional lymphatic specific markers in addition to Prox1. Only when the LEC progenitors fully exit the
cardinal veins do they start to express Podoplanin (PDPN) (18). VEGF-C/VEGFR-3 signaling is also
essential for the sprouting, and deficiency of the pathway results in the failure of lymphatic progenitor
cells to detach themselves from the cardinal vein for further independent lymphatic vasculature
development (20). The lymphatic vasculatures are then formed by sprouting out from the primary lymph
sacs; near birth, lymphatic specific markers are highly expressed and lymphatic branches are fully
differentiated (21).

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1.3 Lymphatics in the eye
A healthy cornea is devoid of any blood and lymphatic vessels, and is transparent to allow light incidence
and refraction. The alymphatic and avascular statuses are maintained by expression of vascular

Figure 3. Lymphatic vessels development. Lymphatic vessels develop from cardinal veins, and the
development is a stepwise process. The expression of Prox1 determine the transition from blood
endothelial cells (BECs) to LEC progenitors. Then the Prox1 expressing cells bud from cardinal vein to form
lymph sacs, and lymph sacs sprout and remodel to form mature lymphatic vessels. Along development, the
lymphatic specific markers gradually express to get fully differentiated and mature LECs. Figure 3 is
modified from Oliver et al., 2010 (15).
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endothelial growth factor (VEGF) receptors and immunoregulatory factors (22). In comparison, the
limbus that surrounds the cornea is filled with lymphatics, which are connected to the conjunctiva, and
only under some pathologic conditions, such as inflammation, infection and trauma, do the lymphatics
in the limbus extend into the cornea. Lymphangiogenesis in the cornea facilitate immune cells trafficking,
so that immune responses are promoted (22). Lymphatics in conjunctiva support metabolism in the eye,
transport immune cells to the anterior part of the eye (23), and remove excess interstitial fluid. It was
viewed that in glaucoma treatment with sclera filter, when there are healthy conjunctival lymphatics in
the area that the filter is placed, the treatment outcomes, i.e. intraocular pressure (IOP) is low (24).
Moreover, the limbal lymphatics were discovered to not be evenly distributed around the circle of limbus;
rather, the nasal side has a greater density of lymphatics, and this nasal dominant pattern is maintained
in response to inflammation (25).

1.4 Comparison between lymphatic and blood vessels in the eye
The development of blood vessels occurs earlier in embryogenesis than lymphatic development. In the
mouse, angiogenesis begins approximately at embryonic day 7.5 (E7.5)(26). Blood vessel endothelial
cells originate from mesodermal cells that express angioblast markers, and during development, the
angioblasts migrate to different places in the embryo and contribute to major vasculatures by
vasculogenesis (27). The blood vessel network is subsequently expanded via angiogenesis, and become
mature and functional by lumen formation and artery-vein differentiation. After artery-vein
differentiation, many signaling pathways drive lymphatic cell differentiation and development, such as
13
Notch signaling, VEGF-A and VEGFR-1 (Flt-1) (27, 28). The molecules that are involved in the two
vasculatures development overlap to some extent – the VEGF family is critical in both processes, though
VEGF-A signaling is more essential in blood vessel development while VEGF-C and VEGF-3 are more
important in lymphatics development (27).

As for the distribution of blood vessels in the eye, there are two distinct yet connected branches deriving
from the ophthalmic artery, one deep and one superficial. Together, these braches provide blood supply
to the eye. The deep branch originates from the long posterior ciliary artery, which goes through the
sclera and form the major circulus anteriorly in the iris. The superficial branch forms the episcleral
arterial circle and is derived from the anterior ciliary arteries. Arterioles branch from the arterial circles
and fill the limbal as well as the conjunctival areas (29) (Figure 4.).

Figure 4. Blood vessel distribution in the eye. The eyeballs have multiple vessel branches for blood supply.
The long posterior ciliary arteries and anterior ciliary arteries branch from ophthalmic artery to supply the
deep and superficial part of the eye respectively with blood.
14

Lymphangiogenesis and angiogenesis always occur concurrently under pathogenic conditions (30), and
a corneal micro-pocket assay showed the interdependency between blood and lymphatic vessels
temporally and spatially. Angiogenesis occurs earlier than lymphangiogenesis, and some growth factors
produced by blood vessels plays a guidance role of lymphatics branching (31). However, angio- and
lymphangiogenesis towards inflammation have different behaviors in the eye: both lymphatic and blood
vessels bear a nasal-dominant under normal conditions in the limbus, but after inflammation, blood
vessels lose this feature while lymphatic vessels maintain it (25). Moreover, another study demonstrated
that preexisting or developing blood vessels in cornea are not essential for lymphangiogenesis, and a
specific dose of fibroblast growth factor 2 (FGF-2) accompanied with VEGF-C and VEGF-D induces only
lymphangiogenesis (32). These evidences shed a question on whether and how the lymphatic and blood
vessels are related with each other.

15

2. Methods and Materials
2.1 Mice and Rats
Prox1-tdtomato and Flt1-tdtomato/ prox1-EGFP reporter mice were used. Flt1 promoter provided blood
vessel specific fluorescence signal whereas Prox1 transcription factor provided a lymphatic specific signal.
The eyes from the reporter mouse pups were processed using the clover method (mentioned in 2.3) and
applied for imaging. For Prox1-tdtomato mice, pups from postnatal day 2 (p2) to postnatal day 10 (p10)
were traced daily, and for Flt1-tdtomato/ prox1-EGFP mice, pups at p1, p3 and p5 were used. Prox1-
EGFP reporter rats were also involved in the experiments, to verify if the results were specific in mice.
The rat eyes were processed with the same clover preparation methods mentioned in 2.3.

2.2 Human sample
Human tissues used for IHC staining were from cadaver eye rims after cornea transplant (Figure 5.). Fresh
tissues were fixed in formalin and embedded in paraffin for sectioning at 4µm thickness. The remaining
cornea tissue was removed so that the limbal area (Figure 5.) was exposed for sectioning.

Figure 5. Schematic of human
sample. Human samples came to our
lab was the leftover tissue after
cornea transplantation (the area
between red circles), which contains
part of the cornea, the limbus (the
green circle area) and part of the
sclera.
16

2.3 Processing of Murine Eyeball
Eyeballs from pups were fixed in 4% PFA at 4 ℃ for 2 to 4 hours after a hole was poked at the nasal side
as an orientation marker to reference direction in the future imaging. The fixed eyeballs were cut in half,
and the front half, including the cornea, limbus and conjunctiva, was further processed into the clover
pattern (Figure 6.). The eye clovers were then flattened on slides and observed under fluorescence
microscopes (Leica and Zeiss).

2.4 Whole mount immunofluorescence (IF) staining
Post-natal 2-day old Prox1-tdtomato pup samples were stained with CD31 antibodies (BD Pharmingen
TM
) to image blood vessels. After fixation, the eye clover tissue was permeabilized with 0.5% Triton X-
100 in PBS under room temperature for 20 minutes and blocked in blocking buffer under room

Figure 6. Eye clover processing. The eyeballs from pups are marked at the nasal side by poking a hole
(location denoted by red “x”). Then the eyeballs were cut into half and the front half, containing the cornea,
limbus, conjunctiva and part of sclera, was further cut and flattened on the slide to make a clover pattern.
17
temperature for 1 hour. The tissue was then incubated in CD31 antibody solution (1:500 dilution) at 4 ℃
overnight. A secondary antibody conjugated with green fluorescence probe was then applied to tissue
for 2 hours at room temperature.

2.5 Immunohistochemistry (IHC) staining
The human corneoscleral rim paraffin sections were first processed with paraffin removal and heat-
induced antigen retrieval steps. Then they were blocked with blocking buffer in room temperature for
40 minutes and incubated overnight with primary antibody (Podoplanin, Cell Marque, 1:100 dilution) at
4 ℃. ImmPRESS secondary antibody was applied to sections in room temperature for 30 minutes and
DAB was used for showing a brown color at the staining sites.

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3. Results
3.1 Conjunctival and limbal lymphatics originate from a single route at the nasal side
Eyeballs from postnatal day 2 (p2) Prox1-tdtomato pups and p14 Prox1-EGFP rats were processed as
described in Methods 2.3 and imaged. Prox1 is a lymphatic endothelial specific transcription factor. Thus,
transgenic mice or rats expressing a fluorescent protein under Prox1 will show fluorescent lymphatics
when imaged. It was found that in both mice and rats, the eye lymphatics originate from one single route,
which is located at the nasal side (Figure 7.). The single origination of lymphatics come to conjunctiva
and then penetrate to the limbus. Before birth, the conjunctival lymphatics have already formed a part,
but limbal lymphatics are barely developed in mice. Both conjunctival and limbal lymphatics bear
postnatal development.
19

3.2 Eye lymphatics develop bidirectionally from the nasal side postnatally
P2 to p10 Prox1-tdtomato pups were traced daily to study the development of the eye lymphatics. It
was observed that the eye lymphatics develop bidirectionally from the nasal side to enclose the cornea
(Figure 8.). From p2 to p4, the eye lymphatics have not enclosed the cornea, and at p2 and p3, the limbal
lymphatics are hardly developed, and at p4, the lymphatic vessels at the limbal area can be observed.
From p5 on, the eye lymphatics make a whole circle that surrounds the cornea. By around p6, the
conjunctival lymphatics are nearly fully developed, while the development of limbal lymphatics
continues. It can be observed that by the end of the tracing, limbal lymphatics develop more branches.

Figure 7. Conjunctival and limbal lymphatics origin from one route at the nasal side. Eyeballs from postnatal
day 2 (p2) Prox1-tdtomato mouse pups (7A) and Prox1-EGFP rat pups (7B) were processed and imaged to
view the origination of the lymphatics in conjunctiva and limbus. It is shown in the image that there is a
single route at the nasal side for lymphatic vessels to branch out (the white asterisks are where the nasal
marker was made).
20

Figure 8. Postnatal development of the conjunctival and limbal lymphatics. The conjunctival and limbal
lymphatics develop bidirectionally from the origination route to enclose the whole cornea postnatally.
Eyeballs from Prox1-tdtomato p2 to p10 pups were imaged, and it can be seen that it is not until p5 do
conjunctival lymphatics make a circle surrounds the cornea. At p2 to p3, the limbal lymphatics are not
clearly shown, and not until around p7 to p8 do limbal lymphatics develop to a circle as well. All white
asterisks refer to the nasal markers.
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Because there is no clear demarcation line between the limbus and conjunctiva, and also because the
limbal and conjunctival lymphatics are connected, it is hard to distinguish limbal lymphatics from
conjunctival lymphatics. To differentiate them, two eyeballs of one pup at p6 were compared (Figure 9.).
One of them was processed routinely, and the conjunctivitis of other one was removed. It is shown that
the most inner circle of the lymphatics is limbal lymphatics, while the outer networks are located in the
conjunctiva.

3.3 Eye blood vessels have different development pattern from lymphatic vessels

Figure 9. Differentiation between limbal and conjunctival lymphatics. Two eyeballs from one p6 pup were
processed to clovers. One of the two eyeballs were removed with the conjunctiva (9A), while the other one
kept it (9B), and then they were imaged. It was viewed that the most inner circle was the limbal lymphatics,
while the outer circles were conjunctival lymphatics.
22
The development of lymphatic and blood vessels were compared in mice eyes as well. Eyeballs from p2
Prox1-tdtamato pups were stained with CD31, a marker for endothelial cells, and green fluorescence
attached secondary antibodies, so that blood vessels show green fluorescence only and lymphatic
vessels show both green and red fluorescence. Compared to lymphatic vessels, blood vessels in the eye
originate from multiple routes, and by p2, they already make a circle surrounding the cornea, and are
distributed to a complex network (Figure 10 A, B & C.). The results were confirmed using a different
mouse model, Flt1-tdtomato/ Prox1-EGFP. Flt1 is a blood vessel specific marker, and in this mouse model,
blood vessels show red fluorescence while lymphatic vessels show green fluorescence. Compared to IF
staining method, this mouse model is more accurate and convenient in showing the lymphatic and blood
vessel networks. Eyeballs from p1, p3 and p5 pups were imaged, and the conclusion was the same (Figure
10 D, E, F, G, H & I.).

23

Figure 10. Different development patterns of lymphatics and blood vessels in the eye. A-C are images of
the eyeball from a p2 Prox1-tdtomato pup. A is the red fluorescence indicating lymphatic vessels, and B is
CD31 IF staining (green fluorescence) indicating both lymphatic and blood vessels. C is the combined image.
D-I are images of the eyeballs from p1, p3 and p5 Flt1-tdtomato/ Prox1-EGFP pups, where blood vessels
show red fluorescence while lymphatic vessels show green. D, F and H are the front view of the whole
eyeball, and E, G and I are images of flattened eye clovers.
24

3.4 Human eye lymphatics distribution is similar to that in mouse models
Human cadaver corneoscleral rims were made into paraffin sections and stained with Podoplanin (PDPN)
to show the lymphatics distribution. The limbal area of the corneoscleral rims were sectioned, and
processed with IHC staining. From the staining, it can be seen that at one side of the eye rim of the limbus,
there is a predominant distribution of lymphatics (Figure 11.), which aligns to the final distribution
pattern of eye lymphatics in mouse models.

Figure 11. Human limbus has polarized distribution of lymphatics. The limbal area of the human
sample was sectioned and stained with PDPN. 11A shows the whole picture of the section and
11B-I are the zoomed in photos of the corresponding dashed rectangles. Black arrowheads point
out the stained lymphatics. at position G, there are obvious lymphatic networks, and at positions
F and G, there are more lymphatics than the other positions. Thus, the lymphatics distribution in
human limbus is also imbalanced, which aligns to the results found in mouse and rat models.
25

4. Discussions
Previous studies such as Ecoiffier et al. have pointed out that in different mouse models, the distribution
of limbal lymphatic vessels was always imbalance, and were distributed more densely at the nasal side.
Moreover, the lymphangiogenesis in response to inflammation was polarized, and the nasal side
maintained the dominant distribution pattern (25). In this study, we illustrate that this nasal-dominant
distribution pattern of lymphatics in the eye could be attributed to their pathway of origin – the eye
lymphatics originate from the nasal side and are distributed in a nasal-dominant fashion from the
beginning. The polarized distribution of eye lymphatics may also apply to lymphatics in the human eye.
Though we were not able to determine orientation from the cadaver tissues, the results from the staining
align well with the conclusions in Ecoffier’s paper and our murine studies, which is that one side of the
eye presents more lymphatics than the other side. Interestingly, there are certain eye diseases, such as
pterygium, that affect the nasal side of the eye more frequently than the temporal side. This
phenomenon might be related to the lymphatic distribution pattern, though the mechanisms are still
largely unknown (25).

Our project also confirms that after the lymphatic endothelial cell fate has been decided and separation
of blood-lymph vasculature has occurred, the maturation and development of lymphatics are
independent from blood vessels spatially and temporally (27). Spatially, murine eye lymphatics originate
from one route at the nasal side, while blood vessels bear multiple routes from both nasal and temporal
26
sides to come into the eye, with no overlap with the lymphatic origination route. The lymphatic and
blood vessels differ temporally in development as well. Eye lymphatics are developed embryonically and
postnatally in our mouse models, while the blood vessels are developed mainly in embryos.

The postnatal development feature of lymphatics is not confined to eyeballs, but also applies to many
other organs; for example, in mouse models, the tail and ear dermis was also well observed to have
postnatal lymphatic development (33). Conjunctiva and limbus could be used as a new model for
studying the signaling pathways and networks that regulate postnatal lymphatic development and
maintenance. Postnatal development and maturation of lymphatic network is critical for the intact
functions of the lymphatics or even survival of the animal model, and it is already known that many
molecules are related with this process. Notch-Dll pathway, besides the primary role of blood vessel
morphogenesis regulation, also regulates postnatal lymphatic development, as Notch signaling is related
to the expression of EphrinB2, which is involved in lymphangiogenic sprouting, and many other
lymphatic specific markers (33). Activin receptor-like kinase-1 (ALK1), which is an essential molecule in
blood vessel development, plays a role in lymphatic development in neonatal mice, and blockade of ALK1
affects differentiation of lymphatic endothelial cells (34). More molecules and pathways are being
revealed to be related to postnatal lymphatic development regulations, some of which are originally
lymphatics development related, while some of which are known molecules that are not lymphatics
development specific with novel functions discovered.

27
The nasal side origination postnatal development fashion of conjunctival and limbal lymphatics may also
affect the outcome of cornea transplant. The statistic from 640 patients aged younger than 20 showed
that the graft survival rate of infants younger than 5 years old is lower than that of other age groups.
Many factors are associated with the outcome of the transplant, including the inflammation,
vascularization and post-graft operative procedures etc. (35). The mechanisms were not fully
understood, but the possibility that the immature lymphatics that are still developing in infants affect
the survival rate cannot be excluded.

28
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Abstract (if available)

Abstract The limbus is a transitional zone between the transparent cornea and the opaque sclera and serves as the surgical incision site for diseases such as cataracts and glaucoma. Current anatomical knowledge indicates that the cornea is devoid of any vasculature while the limbal area is filled with lymphatic vessels, which are connected to the conjunctival lymphatic network. Under inflammatory conditions, the pre-existing lymphatic vessels extend to the cornea, facilitating the exit of antigenic material from the cornea to the lymph nodes to induce an adaptive immune response. It has been shown in adult mice that the nasal side of the limbus bears a predominant pattern of lymphatic distribution

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Asset Metadata

Creator Wu, Yifan (author)

Core Title Development of the lymphatics in the eye

School Keck School of Medicine

Degree Master of Science

Degree Program Biochemistry and Molecular Medicine

Publication Date 01/11/2021

Defense Date 05/02/2019

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

Tag conjunctiva,Development,limbus,lymphatics,OAI-PMH Harvest

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Hong, Young Kwon (committee chair), Lien, Ching Ling (committee member), Zhang-Nunes, Sandy (committee member)

Creator Email wuyifan2017@outlook.com,ywu679@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c89-181900

Unique identifier UC11662462

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Legacy Identifier etd-WuYifan-7538.pdf

Dmrecord 181900

Document Type Thesis

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Rights Wu, Yifan

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...

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