Jagged mediates differences in normal and tumor
angiogenesis by affecting tip-stalk fate decision
Marcelo Boaretoa,b, Mohit Kumar Jollya,c, Eshel Ben-Jacoba,d,1, and Jos N. Onuchica,2
a
Center for Theoretical Biological Physics, Rice University, Houston, TX 77005; bInstitute of Physics, University of Sao Paulo, Sao Paulo 05508, Brazil;
c
Department of Bioengineering, Rice University, Houston, TX 77005; and dSchool of Physics and Astronomy, Tel Aviv University, Tel Aviv 69978, Israel
Contributed by Jos N. Onuchic, June 19, 2015 (sent for review April 30, 2015)
Angiogenesis is critical during development, wound repair, and
cancer progression. During angiogenesis, some endothelial cells
adopt a tip phenotype to lead the formation of new branching
vessels; the trailing stalk cells proliferate to develop the vessel. Notch
and VEGF signaling mediate the selection of these tip endothelial
cells. However, how Jagged, a Notch ligand that is overexpressed in
cancer, affects angiogenesis remains elusive. Here, by developing a
theoretical framework for Notch-Delta-Jagged-VEGF signaling, we
found that higher production levels of Jagged destabilizes the tip
and stalk cell fates and can give rise to a hybrid tip/stalk phenotype
that leads to poorly perfused and chaotic angiogenesis, which is a
hallmark of cancer. Consistently, the signaling interactions that
restrict Notch-Jagged signaling, such as Fringe, cis-inhibition, and in-
creased production of Delta, stabilize tip and stalk fates and limit the
existence of hybrid tip/stalk phenotype. Our results underline how
overexpression of Jagged can transform physiological angiogenesis
into pathological one.
|
angiogenesis Notch signaling | Jagged | VEGF signaling |
tumor angiogenesis
A ngiogenesis, the formation of new blood vessels from existing
ones, is a vital process during embryonic development, ho-
meostasis, and tumor progression (1). This process starts when cells
release angiogenic growth factors such as VEGF in response to
hypoxia (lack of oxygen). These growth factors induce the forma-
tion of a new sprout, and the endothelial cell at the very front of
this angiogenic sprout is called a tip cell. The tip cell extends
numerous filopodia toward the source of these growth factors and
migrates toward the direction of the upward gradient of the growth
factor concentration, thereby leading a new angiogenic branch. The
cells that follow the tip cell do not adopt a tip phenotype, but
rather form the stalk of the branch and proliferate to form the
vessel lumen (2). A well-regulated balance between the migration
of tip cells and proliferation of stalk cells is essential for adequately
shaped nascent sprouts (3).
The selection of the tip and the stalk cell fate is critical for de-
veloping a functional vessel. This decision is mediated by Notch
signaling pathway (2), an evolutionarily conserved cellcell com-
munication pathway involved in cell fate decisions in multiple
contexts. This pathway is activated when Notch (transmembrane
receptor) belonging to a particular cell interacts with Delta or
Jagged (transmembrane ligands) belonging to its neighboring cell
(trans-activation), thereby releasing the Notch intracellular domain
(NICD). NICD then enters the nucleus and modulates the ex-
pression of many target genes of the Notch pathway, including both
the ligands Delta and Jagged. However, when Notch of a cell in-
teracts with Delta or Jagged belonging to the same cell, no NICD is
produced; rather, both the receptor (Notch) and ligand (Delta or
Jagged) are degraded (cis-inhibition) and therefore the signaling is
not activated (4).
Despite generating the same signal (NICD), Notch signaling
activated via Delta and that via Jagged, or in other words, Notch-
Delta (N-D) signaling and Notch-Jagged (N-J) signaling, have dif-
ferent dynamics, because NICD asymmetrically modulates the ex-
pression of the two ligands: it represses Delta but activates Jagged
E3836E3844 | PNAS | Published online July 7, 2015
(5, 6) (Fig. 1A). Therefore, Notch-Delta signaling between two
interacting cells forms an intercellular double negative feedback
loop, and the two cells tend to adopt different fates: one cell be-
haves as a sender [high ligand (Delta), low receptor (Notch)] and
the other one behaves as a receiver [low ligand (Delta), high re-
ceptor (Notch)]. This process of lateral inhibition has a crucial role
in generating a checkerboard-like or salt-and-pepper pattern, as
observed during bristle patterning in flies and inner ear patterning
in vertebrates (7). Conversely, Notch-Jagged signaling generates
an intercellular double positive feedback loop, enabling the two
interacting cells to adopt similar fates: a hybrid sender/receiver [high
ligand (Jagged), high receptor (Notch)] fate. This process of lateral
induction is crucial during sensing development and the formation
of a smooth muscle wall around a nascent artery (6, 8).
Besides asymmetric modulation by NICD, N-D and N-J sig-
naling can also be differentially regulated by glycosyltransferase
Fringe. Fringe modifies Notch such that the modified (or gly-
cosylated) Notch has a higher chance to bind to Delta, but a
lower chance to bind to Jagged (9). Importantly, Fringe, can also
be activated by NICD in some biological contexts (10).
These different dynamics of Notch-Delta and Notch-Jagged sig-
naling allow them to play complementary roles during angiogenesis.
Notch-Delta signaling plays a crucial role in selecting the tip cell in
response to VEGF (11). The binding of VEGF-A (the key ligand of
VEGF family that responds to hypoxia) to VEGF receptor 2
(VEGFR2) (the main mediator of VEGF-A signaling during an-
giogenesis) up-regulates the production of Delta (DLL4) (12).
DLL4 binds to Notch receptor on the neighboring cell and activates
Notch signaling (NICD) in it. NICD inhibits VEGFR2, therefore
Significance
Developing effective antiangiogenesis strategies remains clini-
cally challenging. Unlike physiological angiogenesis, pathological
angiogenesis comprises of many microvessels that do not fully
mature or develop functionally, because the cell fate decision
about which endothelial cells become the tip and lead the fol-
lowing stalk cells is dysregulated. We devised a specific theoret-
ical framework to decipher the cross-talk between two crucial
players of the decision-making process of tip and stalk cell fate:
VEGF and Notch-Delta-Jagged signaling. We find that high ex-
pression of Jagged, but not Delta, can destabilize the terminal
differentiation into tip or stalk cells and give rise to a hybrid tip/
stalk phenotype, a phenotype that can transform physiological
into pathological angiogenesis. Our results offer insights into
why tumor-stroma communication often implicates Jagged.
Author contributions: M.B., M.K.J., E.B.-J., and J.N.O. designed research; M.B. and M.K.J.
performed research; M.B., M.K.J., E.B.-J., and J.N.O. analyzed data; and M.B., M.K.J.,
E.B.-J., and J.N.O. wrote the paper.
The authors declare no conflict of interest.
Freely available online through the PNAS open access option.
1
Deceased June 5, 2015.
2
To whom correspondence should be addressed. Email: jonuchic@rice.edu.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1511814112/-/DCSupplemental.
www.pnas.org/cgi/doi/10.1073/pnas.1511814112
 PNAS PLUS
A B

Fig. 1. Overview of the intracellular and intercellular interplay between Notch and VEGF signaling pathways. (A) Notch signaling is activated when the
transmembrane receptor of one cell (Notch) binds to the transmembrane ligand (Delta or Jagged) of the neighboring cell (trans-activation). This trans-
activation cleaves Notch to produce Notch Intracellular Domain (NICD) that is released in the cytoplasm and then enters the nucleus to modulate the tran-
scription of many target genes. NICD can activate Notch and Jagged and inhibit Delta and VEGF receptor 2 (VEGFR2). Glycosylation of Notch by Fringe
modifies Notch to have a higher affinity for binding to Delta and a lower affinity for binding to Jagged. Interaction between Notch receptor and ligands
(Delta or Jagged) of the same cell (cis-inhibition) leads to the degradation of both the receptor and the ligand; thus, no NICD is generated. VEGF-A binds to
VEGFR2, thus activating VEGF signaling in the cell that activates Delta (DLL4). (B) Cells with high levels of Delta, VEGFR2, and active VEGF signaling develop
filopodia and migrate toward the VEGF-A gradient, leading the formation of the new branch and are called tip cells. DLL4 from tip cells inhibits the
neighboring cells to also adopt a tip phenotype, thereby forcing them to adopt the stalk fate (low Dll4, high Jagged1, and NICD). Stalk cells, by virtue of the
lateral induction characteristics of Notch-Jagged signaling, can induce neighboring cells to adopt a stalk cell, therefore elongating the lumen.

making the adjacent cell less sensitive to the VEGF-A signal (12). lateral inhibition and can be critical for the emergence of a chaotic
The cell with high Delta (and low NICD) becomes the tip, and the blood vessel network as seen during tumor angiogenesis. Finally,
adjacent ones with low levels of Delta (and high NICD) become the we evaluate the role of both Fringe and cis-inhibition in the tip-
stalk (12). This interplay between Notch and VEGF pathways is stalk cell fate decision.
quite tight and dose dependent, i.e., many neighboring cells dy-
namically compete to adopt the tip position but only one of them Results
wins (13). However, unlike other contexts where Notch-Delta The Theoretical Framework. To explore the effects of Jagged in cell
(N-D) signaling leads to salt-and-pepper patterns, i.e., pattern of fate determination during angiogenesis, we generalized our earlier
alternate fates with a wavelength of one cell, in angiogenesis, the theoretical framework of Notch-Delta-Jagged signaling (15) to
two tip cells are usually separated by a few stalk cells, all of which incorporate VEGF signaling. The equations that describe the dy-
have low Delta but high Jagged (Jag1) levels (14). Thus, Notch- namics of Notch (N), Delta (D), Jagged (J), NICD (I), VEGFR2
Jagged (N-J) signaling that regulates lateral induction (6, 8, 15), i.e., (VR), and active VEGF signaling in a cell (V) are
propagation of the same cell fate in adjacent cells, might decide the
distance between two tip cells (Fig. 1B). dN


= N0 H S+ I, I,N N kC D + kT Dext H S I, F,D
Based on these roles of N-D and N-J signaling, it is expected that dt
 [1]
increased production of Jagged would increase the distance be- + kC J + kT Jext H S I, F,J N,
tween two tip cells by reinforcing the lateral induction mechanism
between the stalk cells. However, the available experimental results
dD



are the exact opposite: i.e., higher production rates of Jagged leads = D0 H S I, I,D H S+ V , V ,D D kC H S I, F,D N
to more tip cells (14). Further, one would also expect that in- dt  [2]
creased production of Delta would lead to more tip cells, but as + kT Next D,
experimentally noted, Dll4 acts as a brake on sprouting angio-
genesis (16). These conflicting observations call for an investigation dJ


of the underlying mechanisms of tip and stalk cell-fate selection = J0 H S+ I, I,J J kC H S I, F,J N + kT Next J, [3]
mediated by Notch-Delta-Jagged (N-D-J) signaling. dt
Here, we propose a specific theoretical framework to study the
dI 


interplay between N-D-J and VEGF signaling in the tip-stalk cell = kT N Dext H S I, F,D + Jext H S I, F,J S I, [4]
fate decision during sprouting angiogenesis. We show that cells can dt
attain the stalk position by both lateral inhibition (through high
COMPUTATIONAL BIOLOGY

levels of Delta in the neighboring tip cells) and lateral induction dVR

= VR0 H S I, I,VR kT VR Vext VR , [5]
BIOPHYSICS AND

(through high levels of Jagged in the neighboring stalk cells). dt

However, Delta and Jagged have opposite roles in stabilizing the
tip position: whereas a higher production rate of Jagged makes it
easier for a tip cell to lose its position to a neighboring stalk cell, a
higher production rate of Delta decreases the dynamic competition
between the two cells to adopt the tip position and consequently
stabilizes the tip and stalk cell fates. Our results also suggest the
existence of a hybrid or intermediate tip/stalk phenotype when
Jagged is overexpressed compared with Delta. Cells in this hybrid
tip/stalk fate have compromised migration traits compared with tip
cells; therefore, the vessels led by these cells are expected to be
smaller and poorly perfused compared with those led by the tip
cells. These traits of the hybrid tip/stalk fate enhance dynamic
dV
= kT VR Vext S V , [6]
dt
where represents the degradation rate of N, D, J, VR, and S is the
degradation rate of I and V. N0, D0, J0, and VR0 represent innate
production rates of the Notch, Delta, Jagged, and VEGF receptors,
respectively. Next, Dext, and Jext represent the amounts of external
proteins, i.e., receptor Notch and ligands Delta and Jagged available
from neighboring cells. Similarly, Vext represent the amount of ex-
ternal VEGF. kC represents the cis-inhibition rate, and kT represent
the trans-activation rates of Notch with its ligands (Delta and

Boareto et al. PNAS | Published online July 7, 2015 | E3837

Jagged) and the activation rate of VEGF signaling. H S+ I, I,N and
H S+ I, I,J represent the transcriptional activation of Notch (N)
and Jagged (J) by the signal NICD (I), and H S I, I,D denotes
the repression of Delta (D) by I. H S+ I, I,N , H S+ I, I,J ,
H S I, I,D , and H S I, I,VR are shifted Hill functions. Shifted
Hill functions are defined as H S X, X,Y = H X + X,Y H + X,
where H X is inhibitory Hill function and H + X is excitatory
Hill function, and X,Y denotes the fold change in production of
Y due to X (17, 18). For activation, shifted Hill functions are
depicted by H S+ and > 1; for inhibition, they are depicted by
H S and < 1. = 1 denotes no effect. The effect of Fringe is
considered to increase with the increase of the Notch signal (I)
and is represented by the shifted Hill functions H S I, F,D and
H S I, F,J (15, 19). We considered F,D > 1 and F,J < 1 to repre-
sent Fringe-mediated increase of Notch-Delta (N-D) binding
affinity both for trans- and cis-interactions, and the decrease of
the same for Notch-Jagged (N-J) interactions (20, 21).
The values of the parameters are detailed in SI Appendix,
section S1 and Table S1. The details of model construction are
discussed in SI Appendix, section S2. The models for two inter-
acting cells and many interacting cells are presented in SI Ap-
pendix, sections S3 and S4, respectively. A discussion about the
robustness of the model with respect to changes in parameter
values is presented in SI Appendix, section S5 and Figs. S1 and S2.
The computational analysis was performed in Python using
IPython (22) and PyDSTool (23).
We analyze two cases of the model: (i) single cell driven by
fixed values of external signals: Delta (Dext), Notch (Next), and
Jagged (Jext) representing the amount of proteins in the neigh-
boring cells, and Vext representing the external signal VEGF-A;
and (ii) a multicell system, where cells are coupled with each
other and communicate via the N-D-J signaling in the presence
of an external concentration of VEGF-A signal (Vext).
Notch Mediates Tip-Stalk Fate Decision. To evaluate the basic op-
erating principles of cell fate decision between tip and stalk, we
first analyze the dynamics of Notch-VEGF signaling by consid-
ering one cell in contact with the external signals: Next, Dext, and
Jextparameters that represent the concentration of Notch,
Delta, and Jagged in the neighboring cellsand Vextthe pa-
rameter that represents the amount of external VEGF released
by cells under hypoxia. We find that the circuit is bistable with
two stable states as (i) [high Delta (D), active VEGF signaling
(V) and VEGF receptor (VR), and low NICD (I) and Jagged (J)]
and (ii) [low Delta (D), active VEGF signaling (V) and VEGF
receptor (VR), and high NICD (I) and Jagged (J)]. The former
stable state corresponds to a tip phenotype, and the latter one
corresponds to a stalk (Fig. 2A), i.e., the cell can adopt either of
the two phenotypes: tip or stalk.
Further, we evaluate the different states or phenotypes a cell
can adopt by varying levels of Delta in the neighboring cells
(Dext). For low values of Dext, which mimics the case of neigh-
boring cells being stalk cells, the cell adopts a tip phenotype
(marked by the {tip} phase). Conversely, for high levels of Dext,
which mimics the case of neighboring cell(s) as tip(s), the cell
adopts a stalk phenotype (marked by the {stalk} phase) (Fig. 2B
and SI Appendix, Fig. S3). Interestingly, at intermediate levels of
Dext, we observe a range of bistability, i.e., the cell can be either
tip or stalk (marked by the {tip, stalk} phase) (Fig. 2B and SI
Appendix, Fig. S3), thereby reflecting phenotypic plasticity and
leading to a dynamic lateral inhibition or cell shuffling as ex-
perimentally observed in angiogenesis (13, 24). This region of
bistability{tip, stalk} phaseexists for a large range of values
of external VEGF signal, as long as Dext is at intermediate levels,
thereby indicating the tight coupling of Notch and VEGF sig-
naling in tip-stalk fate decision (Fig. 2C). Similar behavior is
found when varying Jext instead of Dext (SI Appendix, Fig. S4).
E3838 | www.pnas.org/cgi/doi/10.1073/pnas.1511814112
A B
C D
Fig. 2. Nullcline, bifurcation curve, and phase diagrams for the case of a
single cell driven by external proteins Notch, Delta, Jagged, and VEGF.
(A) Nullclines for the case of one cell interacting with fixed levels of external
proteins (Next = Dext = Jext = Vext = 2,000 molecules). Blue nullcline is for the
condition of all ODEs being set to zero except for dI=dt and green nullcline is
for the condition of all ODEs being set to zero except for dD=dt (Eqs. 16).
Unfilled circles represent unstable steady states, whereas red filled circles
represent the two stable states: tip (high Delta, low NICD) and stalk (low
Delta, high NICD). (B) Bifurcation curve of the levels of Delta (D) on the
membrane as a function of the number of external Delta (Dext ). At low Dext ,
the cell adopts the tip fate, whereas at high Dext , the cell adopts the stalk
fate. At intermediate Dext , the cell can adopt either fate: tip or stalk.
(C) Phase diagram (two-parameter bifurcation diagram) as a function of
external Delta (Dext ) and VEGF (Vext ). The monostable phase {tip} corre-
sponds to the state [high Delta (D), VEGF receptor (VR), active VEGF signaling
(V) and low NICD (I) and Jagged (J)], and monostable phase {stalk} corre-
sponds to the state (low D, VR, and V; and high I and J). The bistable phase
{tip, stalk} corresponds to a region of coexistence of both states: tip and
stalk. (D) Phase diagram as a function of external Delta (Dext ) and external
Jagged (Jext ). Bifurcation curves of the levels of VEGF receptor (VR), active
VEGF signaling (V ), NICD (I), and Jagged (J) are included in SI Appendix,
Fig. S3.
Next, we present the phase diagram driven by two control pa-
rametersDext and Jextdenoting the varying conditions for the
different fates of the neighboring cells. We observe that the cells
can attain the stalk fate for both high levels of Dext and Jext, i.e., stalk
cell fate can be obtained by lateral inhibition mediated largely by
Notch-Delta (when neighboring cell is a tip), as well as by lateral
induction mediated largely by Notch-Jagged (when neighboring
cell is a stalk) (Fig. 2D). Therefore, Notch-Jagged signaling can
propagate the stalk cell fate, or in other words, a stalk cell can use
Notch-Jagged signaling to induce its neighboring cells to adopt a
stalk phenotype also. These stalk cells can contribute to lumen
elongation and maintain the required ratio between tip and stalk
cells for developing a functional blood vessel.
Overexpression of Jagged Leads to a Hybrid Tip/Stalk Phenotype.
Next, we investigate the role of Jagged alongside Delta in the
tip-stalk decision making for the one-cell system. We evaluate a
phase diagram as a function of parameters: both the levels of
external Delta (Dext) and the different production levels of the
ligandsJ0 (production rate of Jagged) and D0 (production rate of
Delta). Our results suggest that overexpression of Jagged leads to
the emergence of a previously unidentified phenotype: a hybrid
tip/stalk fate (marked by the {tip/stalk} phase), where the cell
Boareto et al.

expresses intermediate levels of the proteins N, D, J, I, VR, and V
(Fig. 3 AC and SI Appendix, Fig. S5A). A similar hybrid state is
obtained at low production rate of Delta (Fig. 3 DF and SI Ap-
pendix, Fig. S5B), therefore suggesting that the relative production
rate between Delta and Jagged in a cell determines the exis-
tence of this hybrid tip/stalk phenotype (Fig. 3 C and D and SI
Appendix, Fig. S5). Consistently, at a higher production rate of
Delta compared with that of Jagged, the circuit is bistable only,
and therefore the cell can adopt either a tip or a stalk pheno-
type (marked by the {tip, stalk} phase), but not the hybrid tip/
stalk one (Fig. 3 A and F).
We further investigate the dynamics of the circuit for two cells
interacting via N-D-J signaling for different values of the pro-
duction rate of ligands Delta (D0) and Jagged (J0) and fixed
levels of Vext. Our results indicate that at low production rates of
both Delta and Jagged, both cells attain the stalk fate (both cells
in monostable {stalk} phase). However, in the case of a higher
production rate of Delta, one cell adopts the tip position, whereas
the other become a stalk (bistable {tip, stalk} phase), but when the
production rate of Jagged is high, both cells attain the hybrid
tip/stalk phenotype (both cells in monostable {tip/stalk} phase),
thereby being consistent with the canonical role of Notch-Delta in
diversifying cell fates (tip and stalk in this context) and that of
Notch-Jagged in unifying them (the hybrid tip/stalk here) (7, 15,
19) (SI Appendix, Fig. S6).
The hybrid tip/stalk phenotype, obtained under high levels of
Jagged, is reminiscent of and might correspond to the tip-like
thin cytoplasmic projections that extend across the vessel lumen
of the tumor endothelium but not necessarily a nontumor en-
dothelium (25).
Overexpression of Jagged Destabilizes the Tip and Stalk Cell Fates.
To elucidate how overexpression of Jagged affects the stability of
the three different cell fates (tip, stalk, and hybrid tip/stalk), we
represent the phase space of two interacting cells by an effective
potential. The phase space is presented in terms of the levels of
Delta of each cell (D1, D2), such that (high D1, low D2) corre-
sponds to cell 1 as a tip cell and cell 2 as a stalk cell, and vice
PNAS PLUS
versa. The z axis represents the effective potential that is defined
as U = logP, where P = PD1 , D2 is the probability density in
the 2D phase space (D1 D2) (2628). This probability is cal-
culated by using the EulerMaruyama method to approximate
the ordinary differential equation to a stochastic differential
equation that can evaluate the behavior of the cells in the
presence of biological noise. In this representation, a deep basin
of attraction represents that the corresponding cell fate or steady
state is very stable, or in other words, the cell is not likely to
switch its fate to a different one unless under a large amount of
biological noise. Conversely, a shallow basin of attraction facili-
tates a more dynamic fate exchange (plasticity).
Using this representation, we found that a two-cell system
communicating via N-D-J signaling and responding to external
VEGF behaves differently for different values of the production
rates of Jagged (J0). At low production rates of Jagged in both
cells, the system has two stable steady states, both of which com-
prises of one cell in the tip (high levels of Delta) fate or pheno-
type, and the other in stalk (low levels of Delta) phenotype. In one
of these two states, cell 1 is a tip cell, and cell 2 is a stalk cell (high
D1, low D2); and in the other state, its vice versa: cell 2 is a tip cell,
and cell 1 is a stalk cell (low D1, high D2). Both these states have a
deep potential or basin of attraction, suggesting that these cell
fates are very stable and that a large perturbation is required such
that the tip cell (irrespective of whether cell 1 or cell 2 is the tip
cell) loses its position, or in other words, changes its cell fate (Fig.
4A). These results are consistent with previous experimental and
theoretical observations that VEGF-VEGFR-Dll4-Notch-VEGFR
intercellular feedback loop can mediate a stable tip and stalk fate
decision (11, 29), especially under conditions of nonpathological
angiogenesis: low Jag1 and VEGF levels.
However, as Jagged levels in the cells increase due to increased
J0, the potential for these two states becomes increasingly shallow,
and only a small amount of noise can be sufficient to induce a cell
fate transition or exchange, i.e., a tip cell can become a stalk cell
and vice versa (Fig. 4 B and C), hence indicating that high levels of
Jagged in the cells destabilize the tip and stalk cell fates and fa-
cilitate a dynamic competition for the tip position. For very high

A B C

D E F COMPUTATIONAL BIOLOGY
BIOPHYSICS AND

Fig. 3. Dynamical characteristics of the one-cell system for different levels of production rates of the ligands. Bifurcation curves represent the levels of Delta
in response to varying Dext for different production rates of the ligands Delta and Jagged. (A) D0 = 1,000, J0 = 800; (C) D0 = 1,000, J0 = 1,800; (D) D0 = 800,
J0 = 1,400; and (F ) D0 = 1,600, J0 = 1,400 (all units in molecules/h). The phenotype diagrams (center) show the different possible phases when the circuit is
driven by variable levels of external Delta (Dext ), production rate of Delta (D0), and that of Jagged (J0). (B) Phenotype diagram for variable levels of
external Delta (Dext ) and production rate of Jagged (J0). (E) Phenotype diagram for variable levels of external Delta (Dext ) and production rate of Delta (D0).
Bifurcation curve of the levels of VEGF receptor (VR), active VEGF signaling (V), NICD (I), and Jagged (J) for cases C and D are included in SI Appendix, Fig. S5.

Boareto et al. PNAS | Published online July 7, 2015 | E3839


A B
C D
E 2.5 F 2.5
fate exchange rate (h-1)
fate exchange rate (h-1)
2.0
1.5
1.0
0.5
0.0
1000 120020 1400 1600 1800 2000
Jagged production rate (J0) (molec/h)
Fig. 4. 3D representation of the effective potential as a function of Delta in
cell 1 (D1) and in cell 2 (D2). The effective potential is defined as U = logP,
where P = PD1 , D2 is the probability density calculated by solving the differ-
ential equations stochastically using the EulerMaruyama method. A represents
the case of low production rate of Jagged (J0 = 1,000 molecules/h). BD repre-
sent increasingly high production rates of Jagged: J0 = 1,400 molecules/h,
J0 = 1,800 molecules/h, and J0 = 2,200 molecules/h, respectively. (E) Cell fate
exchange rate (a measure of plasticity of the system) for increasing values of
production rates of Jagged (J0). (F) Cell fate exchange rate for increasing values
of production rates of Delta (D0). Red dot represents the standard value as
presented in SI Appendix, Table S1.
levels of Jagged, both cells no longer maintain their distinct tip and
stalk states or phenotypes, but rather adopt the intermediate tip/
stalk state with intermediate levels of Delta (Fig. 4D).
Further, we calculate how the two ligands Delta and Jagged
differently regulate the switching of cell fates between tip and
stalk fates. Experimental observations on dynamic lateral in-
hibition shows that the cell at the tip position is replaced by
another cell in 2 h (13), i.e., the cell fate exchange rate is
around 0.5/h. We first determine the amount of noise in this two-
cell system that can allow a fate exchange rate of 0.5/h (for
D0 = 1,000 and J0 = 1,200 molecules/h; SI Appendix, Table S1)
and then calculate this rate for different values of the production
of Jagged (J0) and Delta (D0), with both cases explored for the
same level of noise as determined earlier. We observed that an
increase in J0 increases this tip position exchange rate (Fig. 4E).
Oppositely, an increase in the production rate of Delta (D0)
significantly decreases the same (Fig. 4F).
JAG1 (Jagged) and DLL4 (Delta) have been reported to play
opposite roles during angiogenesis (14). Thus, unlike high levels
of Jagged, high levels of Delta lead to a lower tip position ex-
change rate and more stable tip and stalk cell fates, therefore
suggesting mutually competing roles of the two ligands in sta-
bilizing the tip and stalk cell fates (Fig. 4F and SI Appendix,
Fig. S7).
E3840 | www.pnas.org/cgi/doi/10.1073/pnas.1511814112
Production Rate of the Two Ligands Regulate Angiogenesis Differently.
Next, we evaluate the dynamics of the circuit at the tissue level, i.e.,
an array of cells interacting via the N-D-J signaling. We considered
the case of a 2D layer of interacting identical cells exposed to a
fixed level of external VEGF (Vext). For low levels of J0 (production
rate of Jagged), there are, on average, more than one stalk cell
between two tip cells, thereby allowing adequate development of
the lumen (that is comprised of stalk cells) and hence a proper and
robust development of the vessel branch: the case of physiological
angiogenesis. However, as the production rate of Jagged (J0) in-
creases, some cells adopt the hybrid tip/stalk phenotype. These
cells, with somewhat compromised tip characteristics, are expected
to develop less filopodia and migrate less than the tip cells, how-
ever, yet initiate a sprout; therefore, the vessels led by these cells
are expected to be relatively smaller and poorly perfused. Thus,
one would expect proper development of vessels but with a higher
vessel density: the case of suboptimal angiogenesis. Last, when
Jagged is overexpressed, most cells can adopt the hybrid tip/stalk
phenotype, leading to an excessive number of small blood vessels
with quite poor perfusion: a case of nonproductive or pathological
angiogenesis as typically observed in cancer (Fig. 5 AC and SI
Appendix, Fig. S8).
2.0
The exact opposite results are observed when varying the pro-
1.5 duction rate of Delta (D0). High and intermediate levels of D0
ensure physiological angiogenesis; but for low levels of D0, the
1.0 number of the hybrid tip/stalk cells increase, thus giving rise to
0.5
many sprouts but a poorly perfused chaotic network, representing
nonproductive or pathological angiogenesis (Fig. 5 A, D, and E).
0.0
800 1000 1200 1400 1600 1800 Our results are consistent with experimental evidence showing that
Delta production rate (D0) (molec/h) deletion or inhibition of DLL4 promotes nonproductive angio-
genesis with poorly perfused vessels (30, 31). It may be noted that
here we do not consider the effect of proliferation of stalk cells and
that of VEGF gradient: two key factors that can alter the number
of tip cells and stalk cells, as well as their spatial distribution.
Interplay Between Notch Signaling and the VEGF Gradient Guides the
Selection of Tip Cell. Besides the production rates of the two ligands,
VEGF gradient has been shown to influence the vascular patterning
(the spatial distribution of the tip and stalk cells) (12). Therefore,
we next incorporate a VEGF gradient in our two-cell system to
evaluate how it alters the relative stability of the different cell fates
the cells attain. Unlike previous cases, now, cell 2 is exposed to a
higher external VEGF signal (Vext) compared with cell 1 (Fig. 6A).
Similar to the earlier case of equal Vext for both cells (Fig. 4B), we
observed two stable steady states: (high D1, low D2) or that cell 1 is
a tip cell and cell 2 is a stalk cell and (low D1, high D2) or that cell 1
is a stalk cell and cell 2 is a tip cell. However, in this case, both these
stable states are not equally stable; rather, the (low D1, high D2)
state is more stable than the (low D2, high D1) state, or in other
words, the cell that receives higher levels of external VEGF signal,
cell 2, is more likely to be the leading tip cell (Fig. 6B). Therefore,
the Notch-VEGF interplay tends to ensure that the leading cell of a
vascular sprout moves in the direction of the upward gradient of
VEGF. We further show that the fate exchange rate decreases with
the increase in steepness of the VEGF gradient, indicating that the
cell that receives higher VEGF signal is more likely to be a tip cell
and maintain its fate (Fig. 6C).
Fringe Stabilizes the Tip and Stalk Cell Fates. Fringe is a glycosyl-
transferase protein that is activated by NICD. It mediates the
posttranslational modifications of Notch and consequently
modulates the binding of Notch to Delta and to Jagged. The
glycosylated (or Fringe-modified) Notch has a higher binding
affinity to Delta but lower binding affinity to Jagged (20, 21). To
evaluate the role of the glycosyltransferase Fringe in the tip-stalk
fate decisions, we calculate the effective potential of a two-cell
system interacting via N-D-J signaling and under the influence of
fixed external VEGF levels. Including the effect of Fringe makes
Boareto et al.
 PNAS PLUS
A

B C

D E

Fig. 5. Patterning at the tissue level. (A) Cartoon representation of physiological, suboptimal, and pathological angiogenesis. In physiological angiogenesis, two
tip cells are separated by a few stalk cells, allowing a proper and robust development of the blood vessel. In the suboptimal case, angiogenesis is increased by a
decrease in the number of stalk cells and the emergence of some hybrid tip/stalk cells that lead to some small blood vessels and poor perfusion. For pathological
angiogenesis, an excessive number of tip/stalk cells lead to a large number of small blood vessels, leading to excessive but nonproductive angiogenesis. (B)
Average of the fraction of cells in (tip), (tip/stalk), or (tip) state as a function of the production of Jagged (J0). (C) Cartoon representation of 1D layer of interacting
cells for increased values of J0. (D) Average of the fraction of cells in (tip), (tip/stalk), or (tip) state as a function of the production of Delta (D0). (E) Cartoon
representation of 1D layer of interacting cells for increased values of D0. The averages were taken over 100 simulations of a 2D layer of 100 100 interacting cells
with a periodic boundary condition. The states of the cells are defined according to the amount of VEGF signal (V): stalk, V < 100; tip/stalk, 100 < V < 300; and tip,
V > 300 molecules. Bidimensional patterning figures representing the levels of V, I, J, and D are presented in SI Appendix, Fig. S8.

the basin of attraction of the two states(high D1, low D2) and
(low D1, high D2)deeper, thereby stabilizing the tip and stalk
fates (Fig. 7 A and B). We further evaluate the effect of Fringe at
the tissue level and show that loss of Fringe leads to an increase
in the number of cells in the hybrid tip/stalk phenotype, thereby
leading to small and poorly perfused blood vessels, typical of
tumor angiogenesis (Fig. 7 C and D). Importantly, this stabili-
zation effect of Fringe is observed even when Fringe is not in-
cluded in the model as a downstream target of NICD, but rather
as an independent variable (SI Appendix, Fig. S9).
These results offer an explanation into why aggressive tumor
types such as basal-like breast cancer often show a loss of
Fringe (3234) and have increased microvessel density (MVD)
and high microvessel proliferation (MVP) compared with the

A B C COMPUTATIONAL BIOLOGY

0.55
Cell #1 Cell #2
BIOPHYSICS AND

0.50
exchange rate (1/h)

0.45

0.40
VEGF-A VEGF-A
0.35

0.30

0.25
1000 1500 2000 2500
Vext in cell #2

Fig. 6. Effect of VEGF gradient on tip and stalk fate decision. (A) Cartoon representation. We simulate two cells interacting via Notch signaling in the presence of a
VEGF gradient: cell 2 receives more VEGF-A signal than cell 1. (B) Effective potential representation for the case of Vext = 1,000 molecules for cell 1 and Vext = 1,500
molecules for cell 2. (C) Fate exchange rate for different values of Vext for cell 2, whereas Vext for cell 1 remains constant (Vext = 1,000 molecules).

Boareto et al. PNAS | Published online July 7, 2015 | E3841

 A B
C D
stalk tip/stalk tip
1.0
0.8
Fraction of cells
0.6
0.4
f Jagged in the stalk cells would reduce the signaling ability of
0.2
0.0
0.0 0.2 0.4 0.6 0.8 1.0
eect of Fringe (f)
Fig. 7. Effect of Fringe on tip and stalk fate decision. (A) 3D representation of
the effective potential as a function of Delta in cell 1 (D1) and in cell 2 (D2) for
the case of no Fringe effect (f = 0.0, i.e., F,D = F,J = 1). B represents the effective
potential after including Fringe effect (f = 1.0, i.e., F,D = 3, F,J = 0.3). The state
with high D2 and low D1, i.e., the one with high levels of Delta in cell 1 but not
in cell 2, corresponds to (cell 1 as tip and cell 2 as stalk); the state with high D1
and low D2 corresponds to (cell 1 as stalk and cell 2 as tip). (C) Average of the
fraction of cells in (stalk), (tip/stalk), or (tip) state as a function of the Fringe
effect. The averages were taken over 100 simulations of a 2D layer of 100 100
interacting cells in a square lattice with periodic boundary conditions. (D) Car-
toon representation of a 1D layer of interacting cells for increased values of the
effect of Fringe. The states of the cells are defined according to the amount
of active VEGF signaling (V): stalk (V < 100), tip/stalk (100 < V < 300), and tip
(V > 300 molecules). The Fringe effect is represented by the variable f. The case
f = 0.0 represents the no Fringe effect, i.e., F,D = F,J = 1, i.e., binding affinity of
Notch to Delta and to Jagged is the same. As f increases, the values of F,D and
F,J linearly increase and decrease, respectively, such that at f = 1.0, F,D = 3.0
and F,J = 0.3 (SI Appendix, Table S1), i.e., Notch has higher binding affinity to
Delta and lower to Jagged. Therefore, (F,D = 1 + 2f) and (F,J = 1 0.7f).
relatively less aggressive ER-positive and HER2-driven sub-
types (35).
cis-Inhibition Stabilizes the Tip and Stalk Cell Fates. cis-Inhibition,
the intracellular binding and consequent degradation of the
Notch receptor and ligands (both Delta and Jagged), has been
considered to be critical for lateral inhibition and pattern for-
mation in multiple developmental contexts (36, 37). However, its
role in angiogenesis remains enigmatic. cis-Inhibition between
Notch and Jagged in the stalk cells has been suggested to com-
promise the tip-to-stalk signaling (14). Thus, we decided to ex-
plore the role of cis-inhibition between Notch and Delta (N-D)
and Notch and Jagged (N-J) both individually and together in
the context of the tip selection process during angiogenesis.
To evaluate the role of cis-inhibition between Notch and both
its ligands Dll4 and Jag1 in angiogenesis, we analyze its effect on
the stability of the tip and stalk cell fates, by representing the
phase space by an effective potential for the case of both lower
and higher cis-inhibition rate (kC). In both cases, two stable
states are present: one cell as tip and the other as stalk and vice
versa [(high D1, low D2), and (low D1, high D2)]. However, at
higher values of kC, the basin of attraction for the stable states
are deeper, therefore suggesting that cis-inhibition has an im-
portant role in stabilizing the tip position (Fig. 8). These results
are consistent with previous experimental and theoretical ob-
servations that cis-inhibition facilitates pattern formation and
usually confers a greater robustness to noise during adoption of
alternate fates between neighboring cells (36, 38, 39).
E3842 | www.pnas.org/cgi/doi/10.1073/pnas.1511814112
We also evaluate the effect of cis-inhibition between Notch-
Delta and Notch-Jagged individually, by changing kC only for
N-D interactions (SI Appendix, Fig. S10 A and B), and then only
for N-J interactions (SI Appendix, Fig. S10 C and D). Our results
suggest that N-J cis-inhibition stabilizes the tip and stalk cell
fates much strongly compared with N-D cis-inhibition, i.e., the
increase in kCJ (cis-inhibition of N-J interactions only) leads to a
much deeper potential well (or basin of attraction) for the two
states(high D1, low D2) and (low D1, high D2)whereas in-
creasing kCD (cis-inhibition of N-D interactions only) has little
effect (SI Appendix, Fig. S10 A and B), again highlighting the fact
that high Jagged levels can destabilize the tip and stalk cell fates
and contribute to the rich cellular plasticity and chaotic behavior
of tumor-mediated angiogenesis.
It has been speculated that cis-inhibition between Notch and
Delta from the tip cell and hence compromise the tip-to-stalk
signaling (14). Our results, however, suggest the opposite, i.e.,
that cis-inhibition has a fundamental role in stabilizing the tip
position. More specifically, we suggest that Notch-Delta cis-
inhibition has relatively little effect in the stability of tip cells,
probably due to the low levels of Notch receptor in the tip cells.
In contrast, Notch-Jagged cis-inhibition has an important role in
stabilizing the tip position, because it decreases the probability of
tip and stalk cells communicating via Notch and Jagged, hence
reducing the levels of NICD in the tip cells. Reduced NICD
implies increased VEGFR2 and consequently high Dll4 in tip
cells, thereby stabilizing the tip cell fate. If N-J cis-inhibition was
low, dynamic competition for tip position would be elevated.
Discussion
Notch and VEGF signaling pathways play a crucial role during
tip-stalk cell fate decisions in both physiological and patho-
logical angiogenesis (1, 12). However, the underlying principles
of tip-stalk fate selection mediated by the interplay of Notch
and VEGF pathways remains largely elusive. Here, we in-
troduced a specific theoretical framework to study this in-
terplay. We show that tip-stalk decision is not a binary one;
rather, cells can adopt a hybrid tip/stalk phenotype, when
Notch-Jagged signaling dominates over Notch-Delta signaling.
This phenotype can lead to form a new sprout but has a com-
promised ability to migrate and develop filopodia, thereby
leading to poorly perfused blood vessels with high MVD.
Therefore, the hybrid tip/stalk phenotype offers a key advan-
tage in pathological conditions: it can confer rich plasticity to
the leading cell that can rapidly exchange its position with a
neighbor stalk, therefore inducing a fast but irregular vessel
Fig. 8. Effect of cis-inhibition on tip and stalk fate decision. (A) 3D repre-
sentation of the effective potential as a function of Delta in cell 1 (D1) and in
cell 2 (D2) for the case of a decrease in 10% of the cis-inhibition strength
compared with its standard value (kC = 4.5e 4). B represents the case of an
increase in 10% of the cis-inhibition strength (kC = 5.5e 4). The state with
high D2 and low D1, i.e., the one with high levels of Delta in cell 1 but not in
cell 2, corresponds to (cell 1 as tip and cell 2 as stalk); that with high D1 and
low D2 corresponds to (cell 1 as stalk and cell 2 as tip).
Boareto et al.

branch that can quickly supply oxygen in fast growing tumors.
When many cells adopt this hybrid phenotype, the vasculature is
expected to be quite chaotic: excessive number of small but
poorly perfused vessels, resulting in pathological angiogenesis as
observed during tumor growth (40). Therefore, our results
offer a good unifying explanation for many experimental ob-
servations: (i) loss of Jagged significantly decreases vascular
branching (14), (ii) loss of Delta leads to excessive non-
productive or poorly perfused angiogenesis (16), and (iii) loss
of Fringe is correlated with increased MVD in tumors (35).
Our results also attempt to resolve an apparent paradox between
the canonical roles of Notch-Delta and Notch-Jagged signaling and
the experimental observations about the overexpression of Delta
and Jagged in angiogenesis. Neighboring cells interacting via Notch-
Jagged signaling usually adopt a similar cell fate (lateral induction)
(6, 8), whereas those interacting via Notch-Delta signaling adopt
opposite fates (lateral inhibition) (7). Consequently, increased
production of Jagged would be expected to reinforce the lateral
induction mechanism between the stalk cells, hence elongating the
lumen; increased production of Delta would lead to more tip cells.
However, the experimental results are the exact opposite: i.e. higher
Jagged levels increase vascular branching (14), and Dll4 acts as a
brake on sprouting angiogenesis (16). These conflicting observa-
tions can be explained by the emergence of a hybrid tip/stalk phe-
notype on overexpression of Jagged. Cells in this hybrid phenotype
can lead the formation of a vessel, albeit not so efficiently, thereby
leading to more vascular branching. Overexpression of Delta can
prevent cells from adopting this hybrid tip/stalk phenotype and can
hence inhibit angiogenesis.
The emergence of a hybrid tip/stalk phenotype also lends sup-
port to the emerging notion a black and white distinction between
tip and stalk cells is an oversimplification (1) and strengthens the
increasingly accepted notion that a hybrid state that coexpresses
markers of two lineages is a signature of enhanced plasticity
(multipotency) of a system (4143). We find that the tendency to
adopt this hybrid phenotype is reduced at high levels of Fringe, a
glycosyltransferase that promotes Notch-Delta signaling at the ex-
pense of Notch-Jagged signaling by modifying Notch to increase its
affinity for Delta and decrease it for Jagged. Thus, Fringe stabilizes
tip and stalk fates and can help promote physiological angiogen-
esis, hence acting as a critical molecular brake on deregulated/
pathological angiogenesis. Loss of this brake, as seen in aggressive
tumors such as basal-like breast cancer (3234), can enable tumors
to attain sustained angiogenesis (35), which is a hallmark of cancer
(44). Overall, our results about Fringe are also consistent with
experimental and theoretical observations that Fringe promotes
lateral inhibition patterns (19, 45) and are reminiscent of how
asymmetric modifications of transmembrane ligand-receptor pairs
can govern tissue-level pattern formation (46).
The importance of Notch-Jagged signaling in delineating the
difference between normal and tumor angiogenesis is further
revealed by the role of cis-inhibition, specifically that between
Notch and Jagged, in affecting tip selection. cis-Inhibition be-
tween Notch and Delta has been reported to offer greater ro-
bustness to noise during patterning (36), but ours is the first
study, to the best of our knowledge, exploring the role of cis-
inhibition between Notch and Jagged. Our results indicate that
cis-inhibition between Notch-Jagged stabilizes tip-and-stalk fates
more strongly than that between Notch-Delta, hence underlining
the role of maintaining low levels of Jagged1 to ensure smooth
and functional, i.e., physiological angiogenesis.
1. Benedito R, Hellstrm M (2013) Notch as a hub for signaling in angiogenesis. Exp Cell
Res 319(9):12811288.
2. Phng LK, Gerhardt H (2009) Angiogenesis: A team effort coordinated by notch. Dev
Cell 16(2):196208.
3. Geudens I, Gerhardt H (2011) Coordinating cell behaviour during blood vessel for-
mation. Development 138(21):45694583.
Boareto et al.
PNAS PLUS
The critical role of overexpression of Jagged1 in mediating such
abnormal angiogenesis might explain why tumor-stroma interplay
often involves Notch-Jagged signaling (47). The increased Notch-
Jagged signaling in tumor environment can be attributed to mul-
tiple specific traits of tumor endothelial cells (TECs): (i) they can
secrete Jagged in the stroma (48) that can potentially activate
Notch-Jagged signaling in neighboring endothelium; (ii) they have
a proinflammatory gene expression and the inflammation re-
sponse regulators such as NF-B and TNF- can increase Jagged
in them (14, 49); and (iii) they often adhere to inflammatory cells
such as macrophages (25) that can increase Jagged in them via
paracrine or juxtacrine signaling. Such an amplified Notch-Jagged
signaling can give rise to hybrid tip/stalk cells that closely resemble
the observed tip-like projections of the tumor vessels that might
overlap with each other and even form loose connections (25).
As discussed here, whereas some predictions of our model are
consistent with reported experimental results, the model offers
some previously untested hypotheses that can be tested experi-
mentally. Specifically, we predict that the interactions that cause
enhanced Notch-Jagged signaling, such as overexpression of
Jag1, repression of Dll4, and inhibition of Fringe, should lead to
a more dynamic switching between tip and stalk cell fates, be-
cause all these cases can cause a larger number of cells to adopt
the hybrid tip/stalk phenotype, hence enriching cellular plasticity.
It might be noted that among the three different homologs of
Fringe in mammals, the role of Lfng (Lunatic fringe) and Manic
fringe (Mfng) might be more pertinent than that of Rfng
(Radical fringe), as they both can promote N-D signaling (50).
To conclude, our theoretical bottom-up modeling framework
offers important insights into the molecular interplay between
Notch and VEGF signaling in regulating cell fate decisions
during both physiological and pathological angiogenesis. Albeit
we do not consider any spatial effects into account, our
framework is amenable to be integrated with agent-based
models on angiogenesis (29) and can be used, in an iterative way
with experiments, to decipher the organizing principles of
multilayer process of angiogenesis (51). Specifically, as re-
ported here, the crucial role of Notch-Jagged signaling in me-
diating differences between physiological and pathological
angiogenesis can be used for novel therapeutic benefits such
as developing decoys that can target JAG/NOTCH selectively
as recently attempted (52). Such attempts are likely to be
more specific in targeting tumor angiogenesis and hence pro-
vide a viable and safer alternative to disrupting Notch signaling
altogether (both via Delta and Jagged), a hallmark of most
antiangiogenesis efforts.
Materials and Methods
The equations for the mathematical model are presented in The Theoretical
Framework. The values of the parameters used for the model are given in SI
Appendix, section S1. Model construction is discussed in SI Appendix, section
S2; and the sensitivity analysis for the model is presented in SI Appendix,
COMPUTATIONAL BIOLOGY
section S5. The computational analysis was performed in Python.
BIOPHYSICS AND
ACKNOWLEDGMENTS. This work was supported by National Science
Foundation Grants PHY-1427654 and NSF-MCB-1214457 and the Cancer
Prevention and Research Institute of Texas. M.B. was also supported by
FAPESP (Sao Paulo Research Foundation) Grant 2013/14438-8. E.B.-J. was
also supported by the Tauber Family Funds and the Maguy-Glass Chair in
Physics of Complex Systems.
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