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SECTION 3: TITLE AND SUMMARY OF NEW PROPOSAL
TITLE: Defining the endothelial-hematopoietic cellular symbiosis
Abstract
Due to their ability to give rise to the whole hematopoietic system, hematopoietic stem and
progenitor cells (HSPCs) have intrigued researchers and physicians alike and their intricate
biology has been a major part of both fundamental and biomedical research activities.
Nevertheless, it is now accepted that HSPCs are far from single functional units, since they need
to reside within a particular microenvironment in the bone marrow that provides them with the
proper chemical, signalling, and cell-to-cell cues for their maintenance, self-renewal, and
proliferation. The ever-growing components of this niche include mesenchymal cells, pericytes,
nerve endings, and endothelial cells (ECs) among others. Although it has been shown that the
endothelium plays a pivotal role in regulating HSPCs biology, so far it has been regarded as a
homogeneous population of cells with similar properties. Taking advantage of novel genetic
tools and highly specific mouse lines, we plan to analyse, with an in vivo level of resolution
previously unattainable, the heterogeneity of the bone marrow endothelium and how each of
the different populations of ECs impacts HSPCs biology. Specifically, we will use several Cre lines
and a new inducible iFUCCI line that allow us to determine simultaneously the location and
specific cell-cycle stage of proliferative endothelial and hematopoietic cells within the bone
marrow. Additionally, we will use other mouse lines when possible to induce and follow the
clonal fate of fluorescently-labelled genetic-cell mosaics with or without specific defects in their
proliferative ability. This will allow us to create a spatiotemporal map of the endothelial
proliferation in the bone marrow and determine the impact of its modulation in the expansion
of adjacent HSPCs. Furthermore, we will also investigate how the HSPCs themselves can impact
the biology of the adjacent endothelium. Finally, to expand our findings to physiologically and
clinically relevant disease models, we will explore if and how the endothelial-hematopoietic
interactions can be relevant for the progression of blood malignancies. This original and
comprehensive research approach will potentially yield novel important insights on the biology
and cross-talk of endothelial and hematopoietic cells which can be later used to design better
therapeutic strategies against related diseases.
AIMS:
1. Use of newly generated genetic tools to visualize and profile in vivo the localization and
genetic program of proliferative endothelial and adjacent hematopoietic cells
2. Mosaic and high-resolution targeting of several genes involved in the blockade or activation
of endothelial cell proliferation - relevance for the adjacent hematopoietic development
3. Targeting the endothelium as a possible alternative way to improve the treatment of
myeloproliferative neoplasms.
Carlos LF de Castillejo Defining the endothelial-hematopoietic symbiosis
1
Defining the endothelial-hematopoietic cellular symbiosis
Abstract
Due to their ability to give rise to the whole hematopoietic system, hematopoietic stem
and progenitor cells (HSPCs) have intrigued researchers and physicians alike and their
intricate biology has been a major part of both fundamental and biomedical research
activities. Nevertheless, it is now accepted that HSPCs are far from single functional
units, since they need to reside within a particular microenvironment in the bone
marrow that provides them with the proper chemical, signalling, and cell-to-cell cues
for their maintenance, self-renewal, and proliferation. The ever-growing components
of this niche include mesenchymal cells, pericytes, nerve endings, and endothelial cells
(ECs) among others. Although it has been shown that the endothelium plays a pivotal
role in regulating HSPCs biology, so far it has been regarded as a homogeneous
population of cells with similar properties. Taking advantage of novel genetic tools and
highly specific mouse lines, we plan to analyse, with an in vivo level of resolution
previously unattainable, the heterogeneity of the bone marrow endothelium and how
each of the different populations of ECs impacts HSPCs biology. Specifically, we will
use several Cre lines and a new inducible iFUCCI line that allow us to determine
simultaneously the location and specific cell-cycle stage of proliferative endothelial and
hematopoietic cells within the bone marrow. Additionally, we will use other mouse
lines when possible to induce and follow the clonal fate of fluorescently-labelled
genetic-cell mosaics with or without specific defects in their proliferative ability. This
will allow us to create a spatiotemporal map of the endothelial proliferation in the bone
marrow and determine the impact of its modulation in the expansion of adjacent
HSPCs. Furthermore, we will also investigate how the HSPCs themselves can impact
the biology of the adjacent endothelium. Finally, to expand our findings to
physiologically and clinically relevant disease models, we will explore if and how the
endothelial-hematopoietic interactions can be relevant for the progression of blood
malignancies. This original and comprehensive research approach will potentially yield
novel important insights on the biology and cross-talk of endothelial and hematopoietic
cells which can be later used to design better therapeutic strategies against related
diseases.
Carlos LF de Castillejo Defining the endothelial-hematopoietic symbiosis
2
Introduction
It is known that endothelial and hematopoietic cells are highly related cell-types with a
common ancestor. Besides having a similar developmental origin, they are also exposed
to the same physical and biological environment. It is also clear that endothelial cells
are an important component of a niche that regulates hematopoietic stem or progenitor
cell biology and function (Morrison and Scadden, 2014). Several groups reported the
close interaction between immunostained CD150+CD48-CD41- cells (HSCs) and
endothelial cells. Conditional deletion of genes like gp130, Vegfr2, Scf and CXCL12
specifically in endothelial cells also led to a reduction in hematopoietic stem cell (HSC)
numbers (Yao et al., 2005; Hooper et al., 2009; Ding and Morrison, 2013; Greenbaum
et al., 2013). In the case of Scf, it was shown that a direct interaction between
membrane-bound endothelial Scf and HSCs is required to maintain their proper
function, suggesting that short-range cues and contact may be involved (Ding et al.,
2012). Besides the close proximity and interaction between endothelial cells and HSCs,
it was also shown recently that most (60-80%) of the immediate progeny of HSCs,
including Sca1+ c-Kit+ cells, as well Lin- c-kit+ multipotent progenitors are also
observed close (< 10um) to the endothelium (Nombela-Arrieta et al., 2013). To date
the endothelial component of the niche has been mainly targeted and seen as a
homogenous entity. However, the diversity in the differentiation and proliferation states
of the different components of the adult bone marrow endothelium and their respective
contributions to HSC biology still remains largely unknown.
Robust expansion and maintenance of long-term HSCs was shown to occur after co-
culture with angiogenic and proliferating endothelial cells, a process that was suggested
to be dependent on the activation by endothelial Notch ligands of canonical receptors
on adjacent HSCs (Butler et al., 2010), similarly to what was proposed to occur during
early development of definitive HSCs (Robert-Moreno et al., 2008). However, at
steady-state conditions, and in the bone marrow, other research groups have shown that
hematopoietic Notch receptor signalling is not required for the maintenance or
differentiation of adult HSCs (Maillard et al., 2008). Importantly, impairment of the
bone marrow endothelial angiogenic status by blocking VEGFR2 and VE-cadherin
signalling significantly reduces reconstitution of LT-HSCs in vivo after sublethal
irradiation (Butler et al., 2010) and endothelial-specific expression of constitutively
active Akt1 accelerates hematopoiesis (Kobayashi et al., 2010). However, is not yet
clear if these observations are linked to an effect of these genes or pathways only on
endothelial proliferation, or on their angiocrine function, even though the latter is
usually proposed to be the mechanism. Impairment of VEGF, AKT or VE-cadherin
signalling has also an effect on many different endothelial processes, such as
endothelial survival, adhesion, permeability, migration, etc. Importantly, in most of
these studies there is no in vivo analysis of the local environment of the targeted
endothelial cells, but rather a general FACS profile of hematopoiesis, which impairs a
direct correlation at the cellular level of direct cause and effect, and the accurate
determination of cell-autonomous vs. non-autonomous effects.
Evidence from different developmental model systems and our own data show that
endothelial proliferation goes hand in hand with the surrounding tissue development,
but technically it is very challenging to distinguish the angiocrine from the tissue
perfusion and nurturing function of blood vessels. Factors secreted by endothelial cells
Carlos LF de Castillejo Defining the endothelial-hematopoietic symbiosis
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in general are known to help the development of surrounding tissues, like during liver
regeneration or bone growth (Hu et al., 2014; Ramasamy et al., 2015), however is not
yet known if proliferating endothelial cells are far more capable of promoting adjacent
tissue development, when compared with adjacent endothelial cells that exited cell-
cycle or are more quiescent in the same vascular area.
The adult bone marrow endothelium is thought to be mostly quiescent, with sporadic
homeostatic proliferation that nonetheless may have other important biological
functions. Our own analyses show that angiogenic or proliferating endothelial cells
express Notch and VEGF receptors at much higher levels than non-proliferating or
quiescent endothelial cells. Our main hypothesis is that within the context of the bone
marrow endothelial niche, HSCs or hematopoietic progenitors are modulated mainly
by a subset of proliferating endothelial cells, and not by the majority of quiescent
endothelial cells. In most experiments done so far, the entire endothelium was targeted,
and not just a subset of endothelial cells. In addition, effects of endothelial cells on
hematopoiesis were scored in the entire tissue and not locally, and never in the context
of mosaic mutant versus control endothelial cells in the same tissue. This was mainly
due to limitations of the genetic tools available. Here we propose to use new mouse
models to target, visualize and understand with great spatial and temporal detail how
quiescent and proliferating endothelial cells impact the development of neighboring
hematopoietic cells and vice-versa. This will certainly increase our understanding of
the bone marrow endothelial niche function and might lead to the development of better
strategies to control the severity of myeloid hypo- or hyperproliferative diseases.
Research Goals
1. Use of newly generated genetic tools to visualize and profile in vivo the
localization and genetic program of proliferative endothelial and adjacent
hematopoietic cells
The host laboratory of my PhD developed in the last years a unique set of tools that
allow the specific genetic manipulation, visualization, and isolation of distinct
endothelial populations in the bone marrow (Table 1). We believe that this will allow us
to characterize the localization and proliferation status of each type of geneticallymodified cell
with unprecedented accuracy. After intercrossing one of the mouse lines we generated
(iFUCCI, Fig. 1), with the Tie2-Cre mouse line (Table 1), we can visualize and distinguish at
the same time 8 different-stages of the endothelial and hematopoietic cell-cycle, by profiling
the relative intensity of the Venus/GFP and Cherry signal (Fig. 1).
The histological localization of hematopoietic stem and progenitor cells (HSPCs) in relation to
the laminin-covered endothelium was already extensively characterized with the use of
powerful confocal imagingand software based quantitative cytometry(Nombela-Arrieta et al.,
2013). We intend to use a similar approach with our iFUCCI mice to perform a
comprehensive quantitative analysis of the localization and cell-cycle stage of HSPCs
and associated endothelial cells, since this allele will be active in both cell types. We
will stain in the same section for several HSPCs markers including Sca1, c-Kit, CD48,
CD150 and CD41 (Nombela-Arrieta et al., 2013), endomucin (endothelial surface), Erg
(endothelial nuclei), Venus/GFP and Cherry (cell-cycle markers). This will allow us to
Carlos LF de Castillejo Defining the endothelial-hematopoietic symbiosis
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see if there is a correlation between endothelial and HSPCs proliferation, in relation to their
relative distance and localization, under homeostatic conditions in vivo.
In parallel, we will also isolate bone marrow endothelial and HSPCs at different stages of the
cell cycle and profile their transcriptomes. This will lead to the identification of genes enriched
in the proliferative fraction of these two populations. Because among the endothelial
proliferative fraction there could be cells that aresporadicallyin s-phase, and cells that received
an induced angiogenic stimulus from the surrounding niche (including HSPCs), we will also
induceandcharacterizetheFUCCIlabelingonlyinthehighlyangiogenic(KdrHigh
orEsm1High
)
fraction of endothelial cells, that can be specifically recombined and detected with the new
Esm1 or Kdr-HA-H2B-Cerulean-2A-iCreERT2 driver mouse lines (Table 1).
Later we will also determine the local endothelial cell-cycle length among Cdh5+ (ALL ECs)
or Kdr/Esm1/Endomucin High
ECs in the different bone marrow regions, by performing low
dose pulse and chase clonal analysis with the combinatorial membrane and nuclear double
mosaic (Fig. 3, mouse line 1 crossed with 2). This will enable us to see how different ECs in
different regions of the bone marrow proliferate over time, and determine if this is the result of
angiogenic or homeostatic EC proliferation. Unlike the iFUCCI cell-cycle stage marker, this
strategy will provide us with a map of long-term endothelial expansion in the bone marrow.
We will also look at the eventual EC heterogeneity within the homeostatic and angiogenic
fractions, by analyzing the distribution and cell-number of the clones. Clones with many cells
arising from a single-cell, surrounded by many individually labeled ECs, would suggest
Carlos LF de Castillejo Defining the endothelial-hematopoietic symbiosis
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differential proliferation/progenitor capacities among a group of ECs, which could be induced
locally by the environment (namely HSPCs). The observation of many clones with an equal
low number of ECs, would on the other hand suggest that all ECs are intrinsically similar in
their proliferative ability in a given location, suggesting that variable cell-to-cell extrinsic cues
do not impact significantly their proliferation.
Carlos LF de Castillejo Defining the endothelial-hematopoietic symbiosis
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In addition to the long-term histological analyses, we will perform another set of experiments
aimed at discerning whether the HSCs exhibit preferential localization in relation to the
surrounding ECs during short-term periods using live imaging microscopy. To test this idea in
vitro, we will first infect normal or immortalized ECs with the iFUCCI virus and at the same
time with an empty control vector or a HA-Cerulean-2A-Oaz1 lentivirus. Oaz1+ endothelial
cells will be quiescent and can be distinguished by their Cherry+ and Cerulean+ fluorescence.
On theother hand, controlECs will be able to go through the cell-cycle in a normal fashion and
the proliferative cells will display Venus/GFP+ fluorescence. Being able to discriminate
proliferative vs. non-proliferative fractions, we will do 24-48 hour co-cultures of ECs with
HSCs labeled with a far-red excitable molecule and examine if the HSCs display an altered
capacity to attach or proliferate on neighboring ECs based on the latter’s proliferative status
(Cerulean+ Cherry+ non-proliferative or Venus/GFP+ proliferative). This strategy will also
allow us to distinguish any chemoattractant or direct niche effect of proliferating ECs over
HSPCs. To strengthen and validate the in vitro data, we will attempt the same experimental
approach with calvarias and long bones of induced iFUCCI mice. Combined, both strategies
will provide us with highly valuable and physiologically relevant information regarding the
spatiotemporal localization of HSCs in respect to the proliferative status of ECs in steady-state
conditions.
To expand our analyses to the clinically relevant situation in which patients might
undergo chemotherapy or radiotherapy prior to bone marrow transplant, we will focus
on the crosstalk between the different types of ECs and HSCs during a short timespan
following a marrow transplant (16 to 24 hours), that crucial period during which the
donor cells enter the bloodstream and lodge within the proper location in the bone
marrow. After injection of a limited number of far-red labelled HSCs into lethally
radiated and induced iFUCCI mice, we will sacrifice the mice 16 to 24 hours after
transplant and measure the distance of each donor HSC in relation to both Cherry+ non-
proliferative and Venus/GFP+ proliferative ECs. The samples will be scanned with a
Zeiss LSM780 two-photon microscope with a MaiThai DeepSee module that allows
the visualization of bone and collagen. Therefore, we will also be able to discriminate
any spatial effects due to factors within the bone-lining endosteal surface (see Figure 2
for an example image). To gain further insights into the role of the endothelium in HSC
homing, live intravital microscopy of mice calvaria will provide us with spatiotemporal
live dynamic information.
Carlos LF de Castillejo Defining the endothelial-hematopoietic symbiosis
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2. Mosaic and high-resolution targeting of several genes involved in the blockade
or activation of endothelial cell proliferation - relevance for the adjacent
hematopoietic development
The ability to conditionally modify the function of genes at any time, or in any tissue,
has changed the way we study biologically and biomedically relevant processes. The
host laboratory developed in the last years new mouse lines that allow the induction of
complex genetic-cell mosaics that enable the study of cellular and clonal phenotypes
with very high spatial and temporal resolution (Fig. 3). With this methodology we will
be able to tell if a given gene acts solely within the cell of that genotype, or if it affects
neighbouring cells, which do not themselves contain that genotype, but take on a
different phenotype due to environmental differentiation. We can also see how a single
genetically modified cell proliferates over a given pulse-and-chase period of time and
how that relates with, or influences, the phenotype of the surrounding environment.
This will be key to understanding the role of the endothelial heterogeneity on
hematopoietic development and vice-versa, in a very complex tissue like the bone
marrow. We will use relatively short-term genetic inductions, and use multicolor
immunostaining and high-resolution confocal imaging on whole-mount thick bone marrow
sections to detect the different endothelial and hematopoietic cell populations (Table 2) in our
control or mutant mice (Table 1). CNIC has 5 confocal microscopes, one of them being a
LEICA SP8 with a white laser that is especially suitable for multispectral excitation and
detection. We can at the moment separate 8 signals of different Alexa-conjugated antibodies
(405, 450, 488, 546, 595, 633, 680, 750) in the same tissue. We also have primary antibodies
produced in unusual species or conjugated with different dyes that allow multicolor detection
in a single immunostaining (Table 2). This gives us the possibility of labeling different
Carlos LF de Castillejo Defining the endothelial-hematopoietic symbiosis
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structures in the same optical slice, significantly improving the speed and information content
of the complex mosaic analysis.
In order to understand the role of endothelial differentiation and proliferation on adjacent
hematopoieticdevelopment we will inducetheloss or gain-of-functionofseveral genes known
to impact the mentioned processes in endothelial cells. We will not start with the use the
classical CreERT2/floxed allele approach due to the lack of reliable markers to identify the
respective genes deletion or gain-of-function with high cellular resolution and in a mosaic
fashion. Instead we will use our recently developed mouse lines where the gain or loss-of-
function of a given gene, or signaling pathway, is directly linked (by the 2A peptide) to the
expression of a membrane or nuclear marker that can be easily detected and separated from
other markers in the same tissue (Fig. 3).
Carlos LF de Castillejo Defining the endothelial-hematopoietic symbiosis
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Due to the pleiotropic nature of many genes, we will manipulate endothelial proliferation by
targeting not only one, but several genes or pathways independently. All the above-mentioned
knock-in alleles (Fig. 3) were already tested and they significantly affect endothelial
proliferation in vivo. The common function of all these genes is the regulation of endothelial
proliferation, even though they control it in different ways and contexts. This will allow us to
characterize how the endothelial proliferation as a whole, and not only a specific genetic
pathway or mechanism, regulates the biology and expansion of adjacent hematopoietic cells.
Later we will alsomanipulate the expression ofsome ofthese genes, like Oaz1andc-Myc, that
are opposing regulators of the cell-cycle, specifically in HSPCs, using the inducible Mx1-Cre
micecrossedwithdifferentconditionallinesliketheMycfloxedandtheOaz1/Odc1 GOFlines
(Table 1), therefore allowing us to examine the potential effect which hematopoietic
proliferation exerts over the adjacent endothelium. In the case that we need to target HSPCs in
a more specific manner, we will infect HSCs with doxycycline-inducible lentivirus. Moreover,
we will attempt to validate and contrast any prospective results by looking at different models
of blood neoplasms that exhibit hyperproliferation of blood cells (see research goal 3).
Integrating the data gathered from the approaches listed above, we will obtain a clearer picture
of the bidirectional symbiosis that occurs between ECs and HSPCs within the marrow.
Carlos LF de Castillejo Defining the endothelial-hematopoietic symbiosis
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3. Targeting the endothelium as a possible alternative way to improve the
treatment of myeloproliferative neoplasms.
Considering the increasing importance that the microenvironment is gaining in
myeloproliferative disease manifestation and progression (García-García et al., 2015),
we plan to target the endothelial component of the niche as a novel therapeutic
opportunity. To induce the disease we will use two murine models of Jak2v617f
neoplasms that have been shown to recapitulate the human malignancy. Mutant JAK2
expression induced by the Vav1-Cre leads to abnormally high platelet production with
slight erythrocythemia, resembling human essential thrombocythemia; while
expression induced by other commonly used driver lines, such as SCL-Cre or Mx1-Cre,
phenocopy aggressive erythrocythemias like human polycythemia vera.
Considering that the role of the endothelium might differ in its magnitude depending
on the stage of disease, we will divide cohorts of mice in two major experimental groups
that will follow a treatment regime that will start either i) 4 weeks after bone marrow
transplant, or ii) after the mice have developed symptoms evident by hematological
counts. In this manner we will be able to distinguish any putative role that ECs may
have on disease manifestation (i) or progression (ii). Mice transplanted with Jak2 v617f
Vav1-Cre
or Jak2 v617f Mx1-Cre
HSPCs mice will receive injections of anti-Vegfr2 (DC101,
Lilly) or anti-Dll4 (YW152F, Genentech) blocking antibodies or control injections of
an IgG vehicle solution. These are supposed to respectively inhibit or induce bone
marrow endothelial proliferation (Ramasamy et al., 2014). We will also use the
inhibitor DFMO (Odc1 inhibitor, Sigma), that is known to block endothelial
proliferation. We might also take advantage of additional ongoing collaborations on the
development of therapeutic antibodies from private companies. Mice will be monitored
with periodical bleeding, and upon sacrifice we will perform FACS analyses to assess
the hematopoietic compartment, and histological analyses to evaluate the extent of
disease in the animals. If successful we will further validate the results gathered from
the use of pharmacological compounds by genetically manipulating the pathways of
interest using the available lines mentioned above (Table 1). The prospective findings
from targeting and better understanding the endothelial niche in myeloproliferative
disease can offer a relevant new insight and approach to treat or ameliorate this disease.
Carlos LF de Castillejo Defining the endothelial-hematopoietic symbiosis
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Tentative work timeline
The following table shows how Dr Benedito and I have plan to achieve the aims listed
in the proposal. Instead of specific months, we have divided each year in 12 segments
to represent each month. Year 3 has not been included, but if an extension is requested,
it will most likely be used to finish any pending revisions of the experiments, solve any
punctual experimental issue, and to finalize any remaining data analyses.
Carlos LF de Castillejo Defining the endothelial-hematopoietic symbiosis
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References
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A.T., Seandel, M., Shido, K., White, I.A., Kobayashi, M., et al. (2010). Endothelial
cells are essential for the self-renewal and repopulation of Notch-dependent
hematopoietic stem cells. Cell Stem Cell 6, 251-264.
Claveria, C., Giovinazzo, G., Sierra, R., and Torres, M. (2013). Myc-driven
endogenous cell competition in the early mammalian embryo. Nature 500, 39-44.
Ding, L. and Morrison, S. J. (2013). Haematopoietic stem cells and early lymphoid
progenitors occupy distinct bone marrow niches. Nature 495, 231–235.
Ding, L., Saunders, T.L., Enikolopov, G., and Morrison, S.J. (2012). Endothelial and
perivascular cells maintain haematopoietic stem cells. Nature 481, 457-462.
García-García, A., LF de Castillejo, C., and Mendez-Ferrer, S. (2015). BMSCs and
hematopoiesis.Immunology Letters.[Epub ahead of print]
Greenbaum, A., Hsu, Y. M., Day, R. B., Schuettpelz, L. G., Christopher, M. J., Borgerding, J.
N., Nagasawa, T., and Link, D. C. (2013). CXCL12 in early mesenchymal progenitors
is required for haematopoietic stem-cell maintenance. Nature 495, 227–230.
Hooper, A. T., Butler, J. M., Nolan, D. J., Kranz, A., Lida, K., Kobayashi, M., Kopp, H. G.,
Shido, K., Petit, I., Yanger, K. et al. (2009). Engraftment and reconstitution of
hematopoiesis is dependent on VEGFR2-mediated regeneration of sinusoidal
endothelial cells. Cell Stem Cell 4, 263–274.
Hu, J., Srivastava, K., Wieland, M., Runge, A., Mogler, C., Besemfelder, E., Terhardt,
D., Vogel, M.J., Cao, L., Korn, C., et al. (2014). Endothelial cell-derived angiopoietin-
2 controls liver regeneration as a spatiotemporal rheostat. Science 343, 416-419.
Kobayashi, H., Butler, J.M., O'Donnell, R., Kobayashi, M., Ding, B.S., Bonner, B.,
Chiu, V.K., Nolan, D.J., Shido, K., Benjamin, L., et al. (2010). Angiocrine factors from
Akt-activated endothelial cells balance self-renewal and differentiation of
haematopoietic stem cells. Nat Cell Biol 12, 1046-1056.
Liu, J., Willet, S.G., Bankaitis, E.D., Xu, Y., Wright, C.V., and Gu, G. (2013). Non-
parallel recombination limits cre-loxP-based reporters as precise indicators of
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J.C., Bhandoola, A., Radtke, F., et al. (2008). Canonical notch signaling is dispensable
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Morrison, S.J., and Scadden, D.T. (2014). The bone marrow niche for haematopoietic
stem cells. Nature 505, 327-334.
Nombela-Arrieta, C., Pivarnik, G., Winkel, B., Canty, K.J., Harley, B., Mahoney,
J.E., Park, S.Y., Lu, J., Protopopov, A., and Silberstein, L.E. (2013). Quantitative
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morphogenesis by endothelial cell-derived signals. Trends Cell Biol 25, 148-157.
Ramasamy, S.K., Kusumbe, A.P., Wang, L., and Adams, R.H. (2014). Endothelial
Notch activity promotes angiogenesis and osteogenesis in bone. Nature 507, 376-380.
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Carlos LF de Castillejo Defining the endothelial-hematopoietic symbiosis
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Defining the endothelial-hematopoietic cellular symbiosis
Cover Letter
The purpose of this application is to ask the Boehringer Ingelheim Fonds for an
extension of my PhD fellowship.
I joined the laboratory of my supervisor, Dr. Rui Benedito at CNIC (Madrid,
Spain) in September 2015. My current PhD fellowship runs from November 1st
, 2015
up until, and including, October 31st
, 2017.
In order to have more time to finalize those aspects of my research aims which
remain to be explored, I would like to ask for an extension to my current PhD fellowship
of 9 months, which would extend the fellowship until and including August 2018.
Regardless of the outcome of the application, I would like to express my
gratitude for the continuing support that I have received from all of the Boehringer
Ingelheim Fonds’ staff.
Thank you,
Carlos López Fernández de Castillejo
Introduction
Here I will very briefly summarize the main ideas and observations from published
research upon which my initial research proposal and the primary objectives to develop
during the course of the fellowship were built.
It has been known for many years now that that i) the biology of endothelial cells and
hematopoietic stem cells are linked to some extent, and ii) the reciprocal effect between
endothelium and hematopoietic progenitors could be due to spatial factors and cues
(reviewed in Morrison and Scadden, 2014).
When looking at the signalling pathways that could be active in both endothelial cells
and HSCs and that could be playing a major role in regulating endothelial and
hematopoietic biology, the Notch signalling pathway emerges as the main candidate
and the main target of my proposal. The understanding of the Notch signalling pathway
and bone marrow biology has seen its framework completely changed due to two recent
paradigm-shifting publications from the Ralf Adams laboratory. Briefly, it was shown
that, contrary to what happens in other organs, in the bone marrow vasculature the
Notch signalling pathway positively regulates angiogenesis (Ramasamy et al., 2014);
in part mediated by the fact that bone marrow vasculature is not spatially homogenous
and can be divided in different vessel subtypes (Kusumbe et al., 2014). Lately, it has
also been suggested that different blood flow patterns in different bone marrow vessels
Carlos LF de Castillejo Defining the endothelial-hematopoietic symbiosis
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can explain the spatial heterogeneity of the bone marrow vasculature (Ramasamy et al.,
2016). From the abovementioned ground-breaking publications, it was shown that loss
of Notch signalling in the bone marrow endothelium caused i) vasculature defects due
to impaired vessel growth and ii) reduced hematopoietic stem cell numbers.
Intriguingly, from our own data we have consistently observed that endothelial-specific
loss of Notch signalling in the bone marrow leads to an increase in endothelial
proliferation (as in other organs), with a concomitant and yet to be described dramatic
increase in the number of hematopoietic progenitors. Moreover, we have observed that
disruption of endothelial Notch signalling in the bone marrow leads to profound
changes in megakaryocyte progenitor numbers. These megakaryocyte progenitors seem
to lie within an interphase between what is currently defined as an endothelial cell and
a hematopoietic progenitor. More striking, nonetheless, we have preliminary evidence
that these megakaryocyte progenitors could have the potential of reconstituting
irradiated mice, therefore behaving as hematopoietic stem cells. This last observation
is in agreement with a recent publication in which researchers demonstrate the existence
of a population of megakaryocyte progenitors with reconstitution capacity in the lung
(Lefrançais et al., 2017).
Following our initial results and observations, and given the potential implications of
refining and modifying the current framework of bone marrow knowledge, and the
novelty of defining a population of megakaryocytes capable of regenerating the
hematopoietic system of irradiated mice, the initial aims of my proposal have been
slightly modified.
Research Goals from the Initial Application
1. Use of newly generated genetic tools to visualize and profile in vivo the
localization and genetic program of proliferative endothelial and adjacent
hematopoietic cells
The idea behind this first aim was to use a mouse line generated by the host laboratory
(iFUCCI, Fig. 1) intercrossed with the endothelial/hematopoietic Tie2-Cre mouse line
in order to visualize and distinguish the different cell-cycle stages of the bone marrow
endothelium and the adjacent hematopoietic cells.
Unfortunately, up until this point in time I have not been able to achieve neither
consistent nor reliable expression of the Venus and Cherry proteins. Although there has
been some cases in which I have observed promising results (Fig. 1), the animals in
which I have made these observations had been sacrificed at the postnatal stage,
therefore not allowing me to have a representative image of the adult bone marrow
vasculature. The host laboratory has continued to develop different strategies to
improve the expression of the Venus and Cherry proteins of iFUCCI line. I will
continue to test these newcoming iFUCCI generations. Given the possibility that I
might not be able to obtain reliable and sufficient samples in the near future, this first
aim of the project will receive a minor part of my focus, which will be directed mostly
towards the completions of the following research aims.
Carlos LF de Castillejo Defining the endothelial-hematopoietic symbiosis
3
Figure 2. Two examples of whole-mount stainings of neonatal femora (animals sacrificed 6 days after
birth) of Tie2-Cre iFUCCI mice. In these cases, the expression of the Venus fluorescent protein is limited
to proliferative cells that are either undergoing S-phase or G2-phase of the cell-cycle, while expression
of the Cherry fluorescent protein labels the non-proliferative cells. Images were acquired with a Zeiss
LSM700 confocal microscope with 10x magnification and 200 micrometers depth.
2. Targeting of several genes involved in the blockade or activation of
endothelial cell Notch signalling - relevance for the adjacent hematopoietic
development
- slightly modified from the initial application-
2(original). Mosaic and high-resolution targeting of several genes involved in the blockade or
activation of endothelial cell proliferation - relevance for the adjacent hematopoietic development
The objective for this research aim in my initial application was to use mouse lines where the
gain or loss-of-function of a given gene, or signaling pathway, is directly linked (by the 2A
peptide) to the expression of a membrane or nuclear marker that can be easily detected and
separated from other markers in the same tissue. Due to the fact that I was not being able to
obtain consistent results and sufficient resolution with the use of inducible genetic mosaics, I
moved from the use of such genetic mosaics and instead decided to use the CreERT2/floxed
allele approach to conditionally eliminate different genes involved in the Notch signaling
pathway.
To pursue this slightly modified research aim, I have used two different endothelial-specific
inducible Cre lines, a Cdh5(PAC)-CreERT2 mouse line (Sorensen et al., 2009) –hereafter
namedVE-Cadherin-CreERT2-, and aPdgfb-CreERT2-IRES-GFP mouse line(Claxton et al.,
2008), intercrossed to RBPj lox/lox and DLL4 lox/lox mice. Both RBPj and DLL4 are key
genes within the Notch signaling pathway, and ablation of either of those alleles with the
abovementioned Cre lines results in endothelial-specific loss of Notch signaling.
All the results obtained from the use of this strategy follow the same schematic outlined in
Figure 3. Adult mice were injected intraperitoneally with 1 mg of tamoxifen for 5 consecutive
days in order to induce the ablation of the gene of interest. 10 days after the first tamoxifen
injection, the mice are sacrificed and the bone marrow phenotype is analyzed by flow
cytometry analyses of hematopoietic and endothelial populations as well as by whole-mount
immunostaining of the vasculature of the femora.
Carlos LF de Castillejo Defining the endothelial-hematopoietic symbiosis
4
Figure 3. Representative diagram of the experimental methodology used to eliminate different Notch-
related genes specifically in the endothelium of adult mice.
Interestingly, and contrary to what is currently accepted to occur in the bone marrow,
it seems that endothelial-specific loss of Notch signalling leads to a drastic increase in
the bone marrow endothelium. We have observed this uncontrolled proliferation of
endothelial cells with the different Cre-lines used, and in some instances, it seems that
this increased proliferation of the endothelium does not stop for at least 10 weeks
(maximum period observed so far). (Fig 4).
Figure 4. Short-term endothelial-
specific loss of Notch signalling in the
adult bone marrow leads to an
increase in the proliferation of
endothelial cells. We have observed
that the deletion of the key effector of the
Notch pathway, RBPj, in the adult
endothelium results in a dramatic
increase in the number of vessels, as
shown here with whole-mount stainings
of femora from adult mice sacrificed 10
days after inducing the loss of RBPj. A-
B: Representative images of the bone
marrow vasculature (labelled with the
vessel markers endomucin and collagen
IV) using two different inducible Cre-
lines. Here we show the results from the
use of these 2 different endothelial-
specific Cre-lines as to demonstrate the
consistency between different models
and to ensure that the effects that we are
observing are not confounded by the
possible genetics inherent different
inducible Cre-lines. As can be observed
with the intensity and abundance of the
vasculature labelling, mice with loss of
Notch signalling present a marked
increase in the vasculature. C: In some of
the experiments in which some mice
were sacrificed after 10 weeks of Notch
loss of function, the increase in
vasculature is still evident, as depicted
here by the increased endomucin
coverage. All images were obtained
using a Zeiss LSM700 confocal
microscope with either 10x or 25x
magnification and with a z-stack of depth
of up to 200 micrometers.
Carlos LF de Castillejo Defining the endothelial-hematopoietic symbiosis
5
These results have been confirmed with flow cytometry analyses of the proliferation
marker Ki67 in combination with the DNA marker Hoescht33342. In all the cases, loss
of endothelial Notch signalling results in increased proportion of proliferative
endothelial cells (Ki67+). (Fig 5.)
Figure 5. Short-term loss of Notch signalling leads to an increase of proliferative endothelial cells.
Endothelial cells from induced mice were isolated and collected using a BD ARIA Cell Sorter and stained
with the proliferation marker Ki67in combination with the DNA marker Hoescht33342. Compared to
the control mice, the bone marrow of mice with loss of RBPj present a higher number of endothelial cells
positive for Ki67.
Additionally, we have observed that short-term specific loss of Notch signalling has
leads to a dramatic increase of hematopoietic progenitor populations. Such dramatic
increase can be linked to an increase proliferative status of such progenitor populations,
as confirmed by Ki67/Hoescht33342 flow cytometry analyses (Fig. 6). One of the major
questions that remain to be answered is whether this increase in progenitor populations
does also lead to changes in their stem-like features, i.e are these progenitors able to
repopulate irradiated mice in the same manner as a wild-type hematopoietic stem cell?
Or does the disruption of endothelial Notch signalling result in altered “stemness” of
hematopoietic stem cells? To answer this I will perform transplants of progenitor cells
that I have stored frozen at -80ºC from all the experiments that I have performed and
compare the ability of such cells to repopulate the irradiated bone marrow and to
regenerate a new hematopoietic system. Finally, I will explore a different hematopoietic
phenotype that has been consistent in all the experiments of Notch loss of function: the
fact loss of Notch signaling leads to very drastic increase in the number of
hematopoietic progenitors present in the peripheral blood (Figure.6C).
Carlos LF de Castillejo Defining the endothelial-hematopoietic symbiosis
6
Figure 6. Short-term loss of Notch signalling leads to several hematopoietic phenotypes. A. Flow
cytometry analyses of hematopoietic progenitor populations reveal a drastic and quite significant
increase in all the progenitor populations when Notch signalling is lost. B. The increase in such
populations can be attributed to a significantly higher proportion of proliferative (Ki67+) progenitor cells
in mice with Notch loss of function. C. Flow cytometry analyses of peripheral blood reveal a massive
increase in the amount of hematopoietic progenitors circulating in the blood in Notch loss of function
mice. For all the experiments, n= 9-12 mice.
From the initial application:
3. Targeting the endothelium as a possible alternative way to improve the treatment
of myeloproliferative neoplasms.
Unfortunately, we had to abandon this aim due to the fact that Dr. Radek Skoda did not
give us his consent to use his published model of Jak2v617f neoplasms. After
deliberation and consideration of the possible alternative models available, we decided
that none of the available mouse models of Jak2v617f neoplasm reliably and
physiologically mimicked the human neoplasms.
3(New). Could megakaryocyte progenitors act as hematopoietic stem cells and
thus be able to repopulate the bone marrow and regenerate the hematopoietic
system of irradiated mice?
Carlos LF de Castillejo Defining the endothelial-hematopoietic symbiosis
7
The pursuit of this newlyproposed aim originates from initial observations and experiments in
which the transplant of megakaryocyte-like cells labelled by the Venus fluorescent protein in
CBF::H2B-Venus(Fig.7A).TheseCBF::H2B-VenusmiceareusedasreportermiceforNotch
activity, i.e the expression of Venus denotes that the Notch signalling pathway is active in
labelled cells. Interestingly, these megakaryocytic cells exhibit expression of both endothelial
and hematopoietic progenitor markers (Fig.7B); which prompted us to test the possibility that
these cells could share features with both of those cells types and therefore could be have the
ability, as with HSCs, to rescue lethally irradiated mice.
Figure 7. Under certain circumstances, megakaryocytic cells could act as hematopoietic stem cells
and therefore be able to regenerate the vascular system of a lethally irradiated adult mouse.
The preliminary results indicate that the transplant of Venus (+) megakaryocyte cells can
indeed rescue irradiated hosts. We have monitored mice transplanted with these
megakaryocyte-like cells for up to 4 months after transplant, and all of the mice in 3 different
transplant experiments not only survived, but showed no vascular abnormalities and displayed
robust engraftment (Fig.7C).
Nevertheless, and thanks to unpublished data from the laboratory, we have also found out that
the expressionof Venus might not necessarilyand accuratelyreflect the activationof the Notch
signalling pathway in a cell. For this reason, we want to repeat the same type of transplant
experiments that have been done with the CBF::H2B-Venus mouse line using instead another
approach. We will use VE-Cadherin-CreERT2 mice intercrossed with a reporter mouse line
R26-tdTomato, which will label endothelial cells and megakaryocytes (unpublished data) by
their expression of the Tomato fluorescent protein. We will then isolate Tomato+
megakaryocytes and transplant them into irradiated hosts in the hopes of replicating our initial
experiments withtheCBF:H2B-Venus line. If successful,we will thentest thesametransplant
setting using instead VE-Cadherin-CreERT2; R26-tdTomato; RBPj lox/lox as donor mice in
order to accurately describe if the repopulating capacity of megakaryocytes is dependent, and
to what extent, upon the Notch signalling pathway.
Carlos LF de Castillejo Defining the endothelial-hematopoietic symbiosis
8
Tentative work timeline
For Aim 1:
I will test newly generated iFUCCI mice in July 2017. If I see improvements in the expression of the
Venus and Cherry fluorescent proteins I will process mice from July 2017 to September 2017 In addition,
I will analyze iFUCCI mice crossed with different Notch-related alleles and repeat the same processing
and quantifications performed with wild-type mice, which would be completed by March 2018. In the
case that the newly generated iFUCCI mice do not meet the expectations for additional processing, I will
have to abandon altogether this aim of the proposal.
For Aim 2:
I am performing a new set of experiments starting in August 2017 in order to collect RNA and bone
samples that I can process for histology. I will perform gene expression analyses between November
2017 and January 2018. I will need to make quantifications of different proliferation markers in thin
paraffin section to confirm all of my preliminary results, which will be performed between November
2017 and March 2018.
For Aim 3:
The mice that I need to serve as donor mice for the transplant will be generated by August 2017. I will
perform the transplant in September 2017 and monitor them for at least 4 months, until the beginning of
January 2018. I will then sacrifice the mice and perform the necessary histology and flow cytometry
analyses, which will be achieved by February 2018. I have set-up crosses to perform similar experiments
with different Notch-related alleles. As the generation of these mice require more than 2 generations, I
will not be able to start this transplants until February 2018. I will then monitor the mice for at least 4
months until May 2018, and will perform the histology and flow cytometry analyses for these transplants,
which will be achieved by August 2018.
References
Claxton, S. et al. (2008). Efficient, inducible Cre-recombinase activation in vascular
endothelium. Genesis 46(2), 74-80.
Kusumbe, A.P., Ramasamy, S.K., and Adams, R.H. (2014). Coupling of angiogenesis and
osteogenesis by a specific vessel subtype in bone. Nature 507, 323-328.
Lefrançais, E., et al.(2017) The lung is a site of platelet biogenesis and a reservoir for
haemotopoetic progenitors. Nature 544, 105-109.
Morrison, S.J., and Scadden, D.T. (2014). The bone marrow niche for haematopoietic
stem cells. Nature 505, 327-334.
Ramasamy, S.K., Kusumbe, A.P., Wang, L., and Adams, R.H. (2014). Endothelial
Notch activity promotes angiogenesis and osteogenesis in bone.
Nature 507, 376-380.
Ramasamy, S.K., et al.(2016). Blood flow control bone vascular function and osteogenesis.
Nature Communications 7, 1-13.
Sorensen, I., et al.(2009). DLL1-mediated Notch activation regulates endothelial identity
in mouse fetal arteries. Blood 113, 5680-5688.

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Project: defining the endothelial hematopoietic symbiosis

  • 1. SECTION 3: TITLE AND SUMMARY OF NEW PROPOSAL TITLE: Defining the endothelial-hematopoietic cellular symbiosis Abstract Due to their ability to give rise to the whole hematopoietic system, hematopoietic stem and progenitor cells (HSPCs) have intrigued researchers and physicians alike and their intricate biology has been a major part of both fundamental and biomedical research activities. Nevertheless, it is now accepted that HSPCs are far from single functional units, since they need to reside within a particular microenvironment in the bone marrow that provides them with the proper chemical, signalling, and cell-to-cell cues for their maintenance, self-renewal, and proliferation. The ever-growing components of this niche include mesenchymal cells, pericytes, nerve endings, and endothelial cells (ECs) among others. Although it has been shown that the endothelium plays a pivotal role in regulating HSPCs biology, so far it has been regarded as a homogeneous population of cells with similar properties. Taking advantage of novel genetic tools and highly specific mouse lines, we plan to analyse, with an in vivo level of resolution previously unattainable, the heterogeneity of the bone marrow endothelium and how each of the different populations of ECs impacts HSPCs biology. Specifically, we will use several Cre lines and a new inducible iFUCCI line that allow us to determine simultaneously the location and specific cell-cycle stage of proliferative endothelial and hematopoietic cells within the bone marrow. Additionally, we will use other mouse lines when possible to induce and follow the clonal fate of fluorescently-labelled genetic-cell mosaics with or without specific defects in their proliferative ability. This will allow us to create a spatiotemporal map of the endothelial proliferation in the bone marrow and determine the impact of its modulation in the expansion of adjacent HSPCs. Furthermore, we will also investigate how the HSPCs themselves can impact the biology of the adjacent endothelium. Finally, to expand our findings to physiologically and clinically relevant disease models, we will explore if and how the endothelial-hematopoietic interactions can be relevant for the progression of blood malignancies. This original and comprehensive research approach will potentially yield novel important insights on the biology and cross-talk of endothelial and hematopoietic cells which can be later used to design better therapeutic strategies against related diseases. AIMS: 1. Use of newly generated genetic tools to visualize and profile in vivo the localization and genetic program of proliferative endothelial and adjacent hematopoietic cells 2. Mosaic and high-resolution targeting of several genes involved in the blockade or activation of endothelial cell proliferation - relevance for the adjacent hematopoietic development 3. Targeting the endothelium as a possible alternative way to improve the treatment of myeloproliferative neoplasms.
  • 2. Carlos LF de Castillejo Defining the endothelial-hematopoietic symbiosis 1 Defining the endothelial-hematopoietic cellular symbiosis Abstract Due to their ability to give rise to the whole hematopoietic system, hematopoietic stem and progenitor cells (HSPCs) have intrigued researchers and physicians alike and their intricate biology has been a major part of both fundamental and biomedical research activities. Nevertheless, it is now accepted that HSPCs are far from single functional units, since they need to reside within a particular microenvironment in the bone marrow that provides them with the proper chemical, signalling, and cell-to-cell cues for their maintenance, self-renewal, and proliferation. The ever-growing components of this niche include mesenchymal cells, pericytes, nerve endings, and endothelial cells (ECs) among others. Although it has been shown that the endothelium plays a pivotal role in regulating HSPCs biology, so far it has been regarded as a homogeneous population of cells with similar properties. Taking advantage of novel genetic tools and highly specific mouse lines, we plan to analyse, with an in vivo level of resolution previously unattainable, the heterogeneity of the bone marrow endothelium and how each of the different populations of ECs impacts HSPCs biology. Specifically, we will use several Cre lines and a new inducible iFUCCI line that allow us to determine simultaneously the location and specific cell-cycle stage of proliferative endothelial and hematopoietic cells within the bone marrow. Additionally, we will use other mouse lines when possible to induce and follow the clonal fate of fluorescently-labelled genetic-cell mosaics with or without specific defects in their proliferative ability. This will allow us to create a spatiotemporal map of the endothelial proliferation in the bone marrow and determine the impact of its modulation in the expansion of adjacent HSPCs. Furthermore, we will also investigate how the HSPCs themselves can impact the biology of the adjacent endothelium. Finally, to expand our findings to physiologically and clinically relevant disease models, we will explore if and how the endothelial-hematopoietic interactions can be relevant for the progression of blood malignancies. This original and comprehensive research approach will potentially yield novel important insights on the biology and cross-talk of endothelial and hematopoietic cells which can be later used to design better therapeutic strategies against related diseases.
  • 3. Carlos LF de Castillejo Defining the endothelial-hematopoietic symbiosis 2 Introduction It is known that endothelial and hematopoietic cells are highly related cell-types with a common ancestor. Besides having a similar developmental origin, they are also exposed to the same physical and biological environment. It is also clear that endothelial cells are an important component of a niche that regulates hematopoietic stem or progenitor cell biology and function (Morrison and Scadden, 2014). Several groups reported the close interaction between immunostained CD150+CD48-CD41- cells (HSCs) and endothelial cells. Conditional deletion of genes like gp130, Vegfr2, Scf and CXCL12 specifically in endothelial cells also led to a reduction in hematopoietic stem cell (HSC) numbers (Yao et al., 2005; Hooper et al., 2009; Ding and Morrison, 2013; Greenbaum et al., 2013). In the case of Scf, it was shown that a direct interaction between membrane-bound endothelial Scf and HSCs is required to maintain their proper function, suggesting that short-range cues and contact may be involved (Ding et al., 2012). Besides the close proximity and interaction between endothelial cells and HSCs, it was also shown recently that most (60-80%) of the immediate progeny of HSCs, including Sca1+ c-Kit+ cells, as well Lin- c-kit+ multipotent progenitors are also observed close (< 10um) to the endothelium (Nombela-Arrieta et al., 2013). To date the endothelial component of the niche has been mainly targeted and seen as a homogenous entity. However, the diversity in the differentiation and proliferation states of the different components of the adult bone marrow endothelium and their respective contributions to HSC biology still remains largely unknown. Robust expansion and maintenance of long-term HSCs was shown to occur after co- culture with angiogenic and proliferating endothelial cells, a process that was suggested to be dependent on the activation by endothelial Notch ligands of canonical receptors on adjacent HSCs (Butler et al., 2010), similarly to what was proposed to occur during early development of definitive HSCs (Robert-Moreno et al., 2008). However, at steady-state conditions, and in the bone marrow, other research groups have shown that hematopoietic Notch receptor signalling is not required for the maintenance or differentiation of adult HSCs (Maillard et al., 2008). Importantly, impairment of the bone marrow endothelial angiogenic status by blocking VEGFR2 and VE-cadherin signalling significantly reduces reconstitution of LT-HSCs in vivo after sublethal irradiation (Butler et al., 2010) and endothelial-specific expression of constitutively active Akt1 accelerates hematopoiesis (Kobayashi et al., 2010). However, is not yet clear if these observations are linked to an effect of these genes or pathways only on endothelial proliferation, or on their angiocrine function, even though the latter is usually proposed to be the mechanism. Impairment of VEGF, AKT or VE-cadherin signalling has also an effect on many different endothelial processes, such as endothelial survival, adhesion, permeability, migration, etc. Importantly, in most of these studies there is no in vivo analysis of the local environment of the targeted endothelial cells, but rather a general FACS profile of hematopoiesis, which impairs a direct correlation at the cellular level of direct cause and effect, and the accurate determination of cell-autonomous vs. non-autonomous effects. Evidence from different developmental model systems and our own data show that endothelial proliferation goes hand in hand with the surrounding tissue development, but technically it is very challenging to distinguish the angiocrine from the tissue perfusion and nurturing function of blood vessels. Factors secreted by endothelial cells
  • 4. Carlos LF de Castillejo Defining the endothelial-hematopoietic symbiosis 3 in general are known to help the development of surrounding tissues, like during liver regeneration or bone growth (Hu et al., 2014; Ramasamy et al., 2015), however is not yet known if proliferating endothelial cells are far more capable of promoting adjacent tissue development, when compared with adjacent endothelial cells that exited cell- cycle or are more quiescent in the same vascular area. The adult bone marrow endothelium is thought to be mostly quiescent, with sporadic homeostatic proliferation that nonetheless may have other important biological functions. Our own analyses show that angiogenic or proliferating endothelial cells express Notch and VEGF receptors at much higher levels than non-proliferating or quiescent endothelial cells. Our main hypothesis is that within the context of the bone marrow endothelial niche, HSCs or hematopoietic progenitors are modulated mainly by a subset of proliferating endothelial cells, and not by the majority of quiescent endothelial cells. In most experiments done so far, the entire endothelium was targeted, and not just a subset of endothelial cells. In addition, effects of endothelial cells on hematopoiesis were scored in the entire tissue and not locally, and never in the context of mosaic mutant versus control endothelial cells in the same tissue. This was mainly due to limitations of the genetic tools available. Here we propose to use new mouse models to target, visualize and understand with great spatial and temporal detail how quiescent and proliferating endothelial cells impact the development of neighboring hematopoietic cells and vice-versa. This will certainly increase our understanding of the bone marrow endothelial niche function and might lead to the development of better strategies to control the severity of myeloid hypo- or hyperproliferative diseases. Research Goals 1. Use of newly generated genetic tools to visualize and profile in vivo the localization and genetic program of proliferative endothelial and adjacent hematopoietic cells The host laboratory of my PhD developed in the last years a unique set of tools that allow the specific genetic manipulation, visualization, and isolation of distinct endothelial populations in the bone marrow (Table 1). We believe that this will allow us to characterize the localization and proliferation status of each type of geneticallymodified cell with unprecedented accuracy. After intercrossing one of the mouse lines we generated (iFUCCI, Fig. 1), with the Tie2-Cre mouse line (Table 1), we can visualize and distinguish at the same time 8 different-stages of the endothelial and hematopoietic cell-cycle, by profiling the relative intensity of the Venus/GFP and Cherry signal (Fig. 1). The histological localization of hematopoietic stem and progenitor cells (HSPCs) in relation to the laminin-covered endothelium was already extensively characterized with the use of powerful confocal imagingand software based quantitative cytometry(Nombela-Arrieta et al., 2013). We intend to use a similar approach with our iFUCCI mice to perform a comprehensive quantitative analysis of the localization and cell-cycle stage of HSPCs and associated endothelial cells, since this allele will be active in both cell types. We will stain in the same section for several HSPCs markers including Sca1, c-Kit, CD48, CD150 and CD41 (Nombela-Arrieta et al., 2013), endomucin (endothelial surface), Erg (endothelial nuclei), Venus/GFP and Cherry (cell-cycle markers). This will allow us to
  • 5. Carlos LF de Castillejo Defining the endothelial-hematopoietic symbiosis 4 see if there is a correlation between endothelial and HSPCs proliferation, in relation to their relative distance and localization, under homeostatic conditions in vivo. In parallel, we will also isolate bone marrow endothelial and HSPCs at different stages of the cell cycle and profile their transcriptomes. This will lead to the identification of genes enriched in the proliferative fraction of these two populations. Because among the endothelial proliferative fraction there could be cells that aresporadicallyin s-phase, and cells that received an induced angiogenic stimulus from the surrounding niche (including HSPCs), we will also induceandcharacterizetheFUCCIlabelingonlyinthehighlyangiogenic(KdrHigh orEsm1High ) fraction of endothelial cells, that can be specifically recombined and detected with the new Esm1 or Kdr-HA-H2B-Cerulean-2A-iCreERT2 driver mouse lines (Table 1). Later we will also determine the local endothelial cell-cycle length among Cdh5+ (ALL ECs) or Kdr/Esm1/Endomucin High ECs in the different bone marrow regions, by performing low dose pulse and chase clonal analysis with the combinatorial membrane and nuclear double mosaic (Fig. 3, mouse line 1 crossed with 2). This will enable us to see how different ECs in different regions of the bone marrow proliferate over time, and determine if this is the result of angiogenic or homeostatic EC proliferation. Unlike the iFUCCI cell-cycle stage marker, this strategy will provide us with a map of long-term endothelial expansion in the bone marrow. We will also look at the eventual EC heterogeneity within the homeostatic and angiogenic fractions, by analyzing the distribution and cell-number of the clones. Clones with many cells arising from a single-cell, surrounded by many individually labeled ECs, would suggest
  • 6. Carlos LF de Castillejo Defining the endothelial-hematopoietic symbiosis 5 differential proliferation/progenitor capacities among a group of ECs, which could be induced locally by the environment (namely HSPCs). The observation of many clones with an equal low number of ECs, would on the other hand suggest that all ECs are intrinsically similar in their proliferative ability in a given location, suggesting that variable cell-to-cell extrinsic cues do not impact significantly their proliferation.
  • 7. Carlos LF de Castillejo Defining the endothelial-hematopoietic symbiosis 6 In addition to the long-term histological analyses, we will perform another set of experiments aimed at discerning whether the HSCs exhibit preferential localization in relation to the surrounding ECs during short-term periods using live imaging microscopy. To test this idea in vitro, we will first infect normal or immortalized ECs with the iFUCCI virus and at the same time with an empty control vector or a HA-Cerulean-2A-Oaz1 lentivirus. Oaz1+ endothelial cells will be quiescent and can be distinguished by their Cherry+ and Cerulean+ fluorescence. On theother hand, controlECs will be able to go through the cell-cycle in a normal fashion and the proliferative cells will display Venus/GFP+ fluorescence. Being able to discriminate proliferative vs. non-proliferative fractions, we will do 24-48 hour co-cultures of ECs with HSCs labeled with a far-red excitable molecule and examine if the HSCs display an altered capacity to attach or proliferate on neighboring ECs based on the latter’s proliferative status (Cerulean+ Cherry+ non-proliferative or Venus/GFP+ proliferative). This strategy will also allow us to distinguish any chemoattractant or direct niche effect of proliferating ECs over HSPCs. To strengthen and validate the in vitro data, we will attempt the same experimental approach with calvarias and long bones of induced iFUCCI mice. Combined, both strategies will provide us with highly valuable and physiologically relevant information regarding the spatiotemporal localization of HSCs in respect to the proliferative status of ECs in steady-state conditions. To expand our analyses to the clinically relevant situation in which patients might undergo chemotherapy or radiotherapy prior to bone marrow transplant, we will focus on the crosstalk between the different types of ECs and HSCs during a short timespan following a marrow transplant (16 to 24 hours), that crucial period during which the donor cells enter the bloodstream and lodge within the proper location in the bone marrow. After injection of a limited number of far-red labelled HSCs into lethally radiated and induced iFUCCI mice, we will sacrifice the mice 16 to 24 hours after transplant and measure the distance of each donor HSC in relation to both Cherry+ non- proliferative and Venus/GFP+ proliferative ECs. The samples will be scanned with a Zeiss LSM780 two-photon microscope with a MaiThai DeepSee module that allows the visualization of bone and collagen. Therefore, we will also be able to discriminate any spatial effects due to factors within the bone-lining endosteal surface (see Figure 2 for an example image). To gain further insights into the role of the endothelium in HSC homing, live intravital microscopy of mice calvaria will provide us with spatiotemporal live dynamic information.
  • 8. Carlos LF de Castillejo Defining the endothelial-hematopoietic symbiosis 7 2. Mosaic and high-resolution targeting of several genes involved in the blockade or activation of endothelial cell proliferation - relevance for the adjacent hematopoietic development The ability to conditionally modify the function of genes at any time, or in any tissue, has changed the way we study biologically and biomedically relevant processes. The host laboratory developed in the last years new mouse lines that allow the induction of complex genetic-cell mosaics that enable the study of cellular and clonal phenotypes with very high spatial and temporal resolution (Fig. 3). With this methodology we will be able to tell if a given gene acts solely within the cell of that genotype, or if it affects neighbouring cells, which do not themselves contain that genotype, but take on a different phenotype due to environmental differentiation. We can also see how a single genetically modified cell proliferates over a given pulse-and-chase period of time and how that relates with, or influences, the phenotype of the surrounding environment. This will be key to understanding the role of the endothelial heterogeneity on hematopoietic development and vice-versa, in a very complex tissue like the bone marrow. We will use relatively short-term genetic inductions, and use multicolor immunostaining and high-resolution confocal imaging on whole-mount thick bone marrow sections to detect the different endothelial and hematopoietic cell populations (Table 2) in our control or mutant mice (Table 1). CNIC has 5 confocal microscopes, one of them being a LEICA SP8 with a white laser that is especially suitable for multispectral excitation and detection. We can at the moment separate 8 signals of different Alexa-conjugated antibodies (405, 450, 488, 546, 595, 633, 680, 750) in the same tissue. We also have primary antibodies produced in unusual species or conjugated with different dyes that allow multicolor detection in a single immunostaining (Table 2). This gives us the possibility of labeling different
  • 9. Carlos LF de Castillejo Defining the endothelial-hematopoietic symbiosis 8 structures in the same optical slice, significantly improving the speed and information content of the complex mosaic analysis. In order to understand the role of endothelial differentiation and proliferation on adjacent hematopoieticdevelopment we will inducetheloss or gain-of-functionofseveral genes known to impact the mentioned processes in endothelial cells. We will not start with the use the classical CreERT2/floxed allele approach due to the lack of reliable markers to identify the respective genes deletion or gain-of-function with high cellular resolution and in a mosaic fashion. Instead we will use our recently developed mouse lines where the gain or loss-of- function of a given gene, or signaling pathway, is directly linked (by the 2A peptide) to the expression of a membrane or nuclear marker that can be easily detected and separated from other markers in the same tissue (Fig. 3).
  • 10. Carlos LF de Castillejo Defining the endothelial-hematopoietic symbiosis 9 Due to the pleiotropic nature of many genes, we will manipulate endothelial proliferation by targeting not only one, but several genes or pathways independently. All the above-mentioned knock-in alleles (Fig. 3) were already tested and they significantly affect endothelial proliferation in vivo. The common function of all these genes is the regulation of endothelial proliferation, even though they control it in different ways and contexts. This will allow us to characterize how the endothelial proliferation as a whole, and not only a specific genetic pathway or mechanism, regulates the biology and expansion of adjacent hematopoietic cells. Later we will alsomanipulate the expression ofsome ofthese genes, like Oaz1andc-Myc, that are opposing regulators of the cell-cycle, specifically in HSPCs, using the inducible Mx1-Cre micecrossedwithdifferentconditionallinesliketheMycfloxedandtheOaz1/Odc1 GOFlines (Table 1), therefore allowing us to examine the potential effect which hematopoietic proliferation exerts over the adjacent endothelium. In the case that we need to target HSPCs in a more specific manner, we will infect HSCs with doxycycline-inducible lentivirus. Moreover, we will attempt to validate and contrast any prospective results by looking at different models of blood neoplasms that exhibit hyperproliferation of blood cells (see research goal 3). Integrating the data gathered from the approaches listed above, we will obtain a clearer picture of the bidirectional symbiosis that occurs between ECs and HSPCs within the marrow.
  • 11. Carlos LF de Castillejo Defining the endothelial-hematopoietic symbiosis 10 3. Targeting the endothelium as a possible alternative way to improve the treatment of myeloproliferative neoplasms. Considering the increasing importance that the microenvironment is gaining in myeloproliferative disease manifestation and progression (García-García et al., 2015), we plan to target the endothelial component of the niche as a novel therapeutic opportunity. To induce the disease we will use two murine models of Jak2v617f neoplasms that have been shown to recapitulate the human malignancy. Mutant JAK2 expression induced by the Vav1-Cre leads to abnormally high platelet production with slight erythrocythemia, resembling human essential thrombocythemia; while expression induced by other commonly used driver lines, such as SCL-Cre or Mx1-Cre, phenocopy aggressive erythrocythemias like human polycythemia vera. Considering that the role of the endothelium might differ in its magnitude depending on the stage of disease, we will divide cohorts of mice in two major experimental groups that will follow a treatment regime that will start either i) 4 weeks after bone marrow transplant, or ii) after the mice have developed symptoms evident by hematological counts. In this manner we will be able to distinguish any putative role that ECs may have on disease manifestation (i) or progression (ii). Mice transplanted with Jak2 v617f Vav1-Cre or Jak2 v617f Mx1-Cre HSPCs mice will receive injections of anti-Vegfr2 (DC101, Lilly) or anti-Dll4 (YW152F, Genentech) blocking antibodies or control injections of an IgG vehicle solution. These are supposed to respectively inhibit or induce bone marrow endothelial proliferation (Ramasamy et al., 2014). We will also use the inhibitor DFMO (Odc1 inhibitor, Sigma), that is known to block endothelial proliferation. We might also take advantage of additional ongoing collaborations on the development of therapeutic antibodies from private companies. Mice will be monitored with periodical bleeding, and upon sacrifice we will perform FACS analyses to assess the hematopoietic compartment, and histological analyses to evaluate the extent of disease in the animals. If successful we will further validate the results gathered from the use of pharmacological compounds by genetically manipulating the pathways of interest using the available lines mentioned above (Table 1). The prospective findings from targeting and better understanding the endothelial niche in myeloproliferative disease can offer a relevant new insight and approach to treat or ameliorate this disease.
  • 12. Carlos LF de Castillejo Defining the endothelial-hematopoietic symbiosis 11 Tentative work timeline The following table shows how Dr Benedito and I have plan to achieve the aims listed in the proposal. Instead of specific months, we have divided each year in 12 segments to represent each month. Year 3 has not been included, but if an extension is requested, it will most likely be used to finish any pending revisions of the experiments, solve any punctual experimental issue, and to finalize any remaining data analyses.
  • 13. Carlos LF de Castillejo Defining the endothelial-hematopoietic symbiosis 12 References Butler, J.M., Nolan, D.J., Vertes, E.L., Varnum-Finney, B., Kobayashi, H., Hooper, A.T., Seandel, M., Shido, K., White, I.A., Kobayashi, M., et al. (2010). Endothelial cells are essential for the self-renewal and repopulation of Notch-dependent hematopoietic stem cells. Cell Stem Cell 6, 251-264. Claveria, C., Giovinazzo, G., Sierra, R., and Torres, M. (2013). Myc-driven endogenous cell competition in the early mammalian embryo. Nature 500, 39-44. Ding, L. and Morrison, S. J. (2013). Haematopoietic stem cells and early lymphoid progenitors occupy distinct bone marrow niches. Nature 495, 231–235. Ding, L., Saunders, T.L., Enikolopov, G., and Morrison, S.J. (2012). Endothelial and perivascular cells maintain haematopoietic stem cells. Nature 481, 457-462. García-García, A., LF de Castillejo, C., and Mendez-Ferrer, S. (2015). BMSCs and hematopoiesis.Immunology Letters.[Epub ahead of print] Greenbaum, A., Hsu, Y. M., Day, R. B., Schuettpelz, L. G., Christopher, M. J., Borgerding, J. N., Nagasawa, T., and Link, D. C. (2013). CXCL12 in early mesenchymal progenitors is required for haematopoietic stem-cell maintenance. Nature 495, 227–230. Hooper, A. T., Butler, J. M., Nolan, D. J., Kranz, A., Lida, K., Kobayashi, M., Kopp, H. G., Shido, K., Petit, I., Yanger, K. et al. (2009). Engraftment and reconstitution of hematopoiesis is dependent on VEGFR2-mediated regeneration of sinusoidal endothelial cells. Cell Stem Cell 4, 263–274. Hu, J., Srivastava, K., Wieland, M., Runge, A., Mogler, C., Besemfelder, E., Terhardt, D., Vogel, M.J., Cao, L., Korn, C., et al. (2014). Endothelial cell-derived angiopoietin- 2 controls liver regeneration as a spatiotemporal rheostat. Science 343, 416-419. Kobayashi, H., Butler, J.M., O'Donnell, R., Kobayashi, M., Ding, B.S., Bonner, B., Chiu, V.K., Nolan, D.J., Shido, K., Benjamin, L., et al. (2010). Angiocrine factors from Akt-activated endothelial cells balance self-renewal and differentiation of haematopoietic stem cells. Nat Cell Biol 12, 1046-1056. Liu, J., Willet, S.G., Bankaitis, E.D., Xu, Y., Wright, C.V., and Gu, G. (2013). Non- parallel recombination limits cre-loxP-based reporters as precise indicators of conditional genetic manipulation. Genesis 51, 436-442. Maillard, I., Koch, U., Dumortier, A., Shestova, O., Xu, L., Sai, H., Pross, S.E., Aster, J.C., Bhandoola, A., Radtke, F., et al. (2008). Canonical notch signaling is dispensable for the maintenance of adult hematopoietic stem cells. Cell Stem Cell 2, 356-366. Morrison, S.J., and Scadden, D.T. (2014). The bone marrow niche for haematopoietic stem cells. Nature 505, 327-334. Nombela-Arrieta, C., Pivarnik, G., Winkel, B., Canty, K.J., Harley, B., Mahoney, J.E., Park, S.Y., Lu, J., Protopopov, A., and Silberstein, L.E. (2013). Quantitative imaging of haematopoietic stem and progenitor cell localization and hypoxic status in the bone marrow microenvironment. Nat Cell Biol 15, 533-543.
  • 14. Carlos LF de Castillejo Defining the endothelial-hematopoietic symbiosis 13 Ramasamy, S.K., Kusumbe, A.P., and Adams, R.H. (2015). Regulation of tissue morphogenesis by endothelial cell-derived signals. Trends Cell Biol 25, 148-157. Ramasamy, S.K., Kusumbe, A.P., Wang, L., and Adams, R.H. (2014). Endothelial Notch activity promotes angiogenesis and osteogenesis in bone. Nature 507, 376-380. Robert-Moreno, A., Guiu, J., Ruiz-Herguido, C., Lopez, M.E., Ingles-Esteve, J., Riera, L., Tipping, A., Enver, T., Dzierzak, E., Gridley, T., et al. (2008). Impaired embryonic haematopoiesis yet normal arterial development in the absence of the Notch ligand Jagged1. EMBO J 27, 1886-1895. Yao, L., Yokota, T., Xia, L., Kincade, P. W. and McEver, R. P. (2005). Bone marrow dysfunction in mice lacking the cytokine receptor gp130 in endothelial cells. Blood 106, 4093–4101.
  • 15. Carlos LF de Castillejo Defining the endothelial-hematopoietic symbiosis 1 Defining the endothelial-hematopoietic cellular symbiosis Cover Letter The purpose of this application is to ask the Boehringer Ingelheim Fonds for an extension of my PhD fellowship. I joined the laboratory of my supervisor, Dr. Rui Benedito at CNIC (Madrid, Spain) in September 2015. My current PhD fellowship runs from November 1st , 2015 up until, and including, October 31st , 2017. In order to have more time to finalize those aspects of my research aims which remain to be explored, I would like to ask for an extension to my current PhD fellowship of 9 months, which would extend the fellowship until and including August 2018. Regardless of the outcome of the application, I would like to express my gratitude for the continuing support that I have received from all of the Boehringer Ingelheim Fonds’ staff. Thank you, Carlos López Fernández de Castillejo Introduction Here I will very briefly summarize the main ideas and observations from published research upon which my initial research proposal and the primary objectives to develop during the course of the fellowship were built. It has been known for many years now that that i) the biology of endothelial cells and hematopoietic stem cells are linked to some extent, and ii) the reciprocal effect between endothelium and hematopoietic progenitors could be due to spatial factors and cues (reviewed in Morrison and Scadden, 2014). When looking at the signalling pathways that could be active in both endothelial cells and HSCs and that could be playing a major role in regulating endothelial and hematopoietic biology, the Notch signalling pathway emerges as the main candidate and the main target of my proposal. The understanding of the Notch signalling pathway and bone marrow biology has seen its framework completely changed due to two recent paradigm-shifting publications from the Ralf Adams laboratory. Briefly, it was shown that, contrary to what happens in other organs, in the bone marrow vasculature the Notch signalling pathway positively regulates angiogenesis (Ramasamy et al., 2014); in part mediated by the fact that bone marrow vasculature is not spatially homogenous and can be divided in different vessel subtypes (Kusumbe et al., 2014). Lately, it has also been suggested that different blood flow patterns in different bone marrow vessels
  • 16. Carlos LF de Castillejo Defining the endothelial-hematopoietic symbiosis 2 can explain the spatial heterogeneity of the bone marrow vasculature (Ramasamy et al., 2016). From the abovementioned ground-breaking publications, it was shown that loss of Notch signalling in the bone marrow endothelium caused i) vasculature defects due to impaired vessel growth and ii) reduced hematopoietic stem cell numbers. Intriguingly, from our own data we have consistently observed that endothelial-specific loss of Notch signalling in the bone marrow leads to an increase in endothelial proliferation (as in other organs), with a concomitant and yet to be described dramatic increase in the number of hematopoietic progenitors. Moreover, we have observed that disruption of endothelial Notch signalling in the bone marrow leads to profound changes in megakaryocyte progenitor numbers. These megakaryocyte progenitors seem to lie within an interphase between what is currently defined as an endothelial cell and a hematopoietic progenitor. More striking, nonetheless, we have preliminary evidence that these megakaryocyte progenitors could have the potential of reconstituting irradiated mice, therefore behaving as hematopoietic stem cells. This last observation is in agreement with a recent publication in which researchers demonstrate the existence of a population of megakaryocyte progenitors with reconstitution capacity in the lung (Lefrançais et al., 2017). Following our initial results and observations, and given the potential implications of refining and modifying the current framework of bone marrow knowledge, and the novelty of defining a population of megakaryocytes capable of regenerating the hematopoietic system of irradiated mice, the initial aims of my proposal have been slightly modified. Research Goals from the Initial Application 1. Use of newly generated genetic tools to visualize and profile in vivo the localization and genetic program of proliferative endothelial and adjacent hematopoietic cells The idea behind this first aim was to use a mouse line generated by the host laboratory (iFUCCI, Fig. 1) intercrossed with the endothelial/hematopoietic Tie2-Cre mouse line in order to visualize and distinguish the different cell-cycle stages of the bone marrow endothelium and the adjacent hematopoietic cells. Unfortunately, up until this point in time I have not been able to achieve neither consistent nor reliable expression of the Venus and Cherry proteins. Although there has been some cases in which I have observed promising results (Fig. 1), the animals in which I have made these observations had been sacrificed at the postnatal stage, therefore not allowing me to have a representative image of the adult bone marrow vasculature. The host laboratory has continued to develop different strategies to improve the expression of the Venus and Cherry proteins of iFUCCI line. I will continue to test these newcoming iFUCCI generations. Given the possibility that I might not be able to obtain reliable and sufficient samples in the near future, this first aim of the project will receive a minor part of my focus, which will be directed mostly towards the completions of the following research aims.
  • 17. Carlos LF de Castillejo Defining the endothelial-hematopoietic symbiosis 3 Figure 2. Two examples of whole-mount stainings of neonatal femora (animals sacrificed 6 days after birth) of Tie2-Cre iFUCCI mice. In these cases, the expression of the Venus fluorescent protein is limited to proliferative cells that are either undergoing S-phase or G2-phase of the cell-cycle, while expression of the Cherry fluorescent protein labels the non-proliferative cells. Images were acquired with a Zeiss LSM700 confocal microscope with 10x magnification and 200 micrometers depth. 2. Targeting of several genes involved in the blockade or activation of endothelial cell Notch signalling - relevance for the adjacent hematopoietic development - slightly modified from the initial application- 2(original). Mosaic and high-resolution targeting of several genes involved in the blockade or activation of endothelial cell proliferation - relevance for the adjacent hematopoietic development The objective for this research aim in my initial application was to use mouse lines where the gain or loss-of-function of a given gene, or signaling pathway, is directly linked (by the 2A peptide) to the expression of a membrane or nuclear marker that can be easily detected and separated from other markers in the same tissue. Due to the fact that I was not being able to obtain consistent results and sufficient resolution with the use of inducible genetic mosaics, I moved from the use of such genetic mosaics and instead decided to use the CreERT2/floxed allele approach to conditionally eliminate different genes involved in the Notch signaling pathway. To pursue this slightly modified research aim, I have used two different endothelial-specific inducible Cre lines, a Cdh5(PAC)-CreERT2 mouse line (Sorensen et al., 2009) –hereafter namedVE-Cadherin-CreERT2-, and aPdgfb-CreERT2-IRES-GFP mouse line(Claxton et al., 2008), intercrossed to RBPj lox/lox and DLL4 lox/lox mice. Both RBPj and DLL4 are key genes within the Notch signaling pathway, and ablation of either of those alleles with the abovementioned Cre lines results in endothelial-specific loss of Notch signaling. All the results obtained from the use of this strategy follow the same schematic outlined in Figure 3. Adult mice were injected intraperitoneally with 1 mg of tamoxifen for 5 consecutive days in order to induce the ablation of the gene of interest. 10 days after the first tamoxifen injection, the mice are sacrificed and the bone marrow phenotype is analyzed by flow cytometry analyses of hematopoietic and endothelial populations as well as by whole-mount immunostaining of the vasculature of the femora.
  • 18. Carlos LF de Castillejo Defining the endothelial-hematopoietic symbiosis 4 Figure 3. Representative diagram of the experimental methodology used to eliminate different Notch- related genes specifically in the endothelium of adult mice. Interestingly, and contrary to what is currently accepted to occur in the bone marrow, it seems that endothelial-specific loss of Notch signalling leads to a drastic increase in the bone marrow endothelium. We have observed this uncontrolled proliferation of endothelial cells with the different Cre-lines used, and in some instances, it seems that this increased proliferation of the endothelium does not stop for at least 10 weeks (maximum period observed so far). (Fig 4). Figure 4. Short-term endothelial- specific loss of Notch signalling in the adult bone marrow leads to an increase in the proliferation of endothelial cells. We have observed that the deletion of the key effector of the Notch pathway, RBPj, in the adult endothelium results in a dramatic increase in the number of vessels, as shown here with whole-mount stainings of femora from adult mice sacrificed 10 days after inducing the loss of RBPj. A- B: Representative images of the bone marrow vasculature (labelled with the vessel markers endomucin and collagen IV) using two different inducible Cre- lines. Here we show the results from the use of these 2 different endothelial- specific Cre-lines as to demonstrate the consistency between different models and to ensure that the effects that we are observing are not confounded by the possible genetics inherent different inducible Cre-lines. As can be observed with the intensity and abundance of the vasculature labelling, mice with loss of Notch signalling present a marked increase in the vasculature. C: In some of the experiments in which some mice were sacrificed after 10 weeks of Notch loss of function, the increase in vasculature is still evident, as depicted here by the increased endomucin coverage. All images were obtained using a Zeiss LSM700 confocal microscope with either 10x or 25x magnification and with a z-stack of depth of up to 200 micrometers.
  • 19. Carlos LF de Castillejo Defining the endothelial-hematopoietic symbiosis 5 These results have been confirmed with flow cytometry analyses of the proliferation marker Ki67 in combination with the DNA marker Hoescht33342. In all the cases, loss of endothelial Notch signalling results in increased proportion of proliferative endothelial cells (Ki67+). (Fig 5.) Figure 5. Short-term loss of Notch signalling leads to an increase of proliferative endothelial cells. Endothelial cells from induced mice were isolated and collected using a BD ARIA Cell Sorter and stained with the proliferation marker Ki67in combination with the DNA marker Hoescht33342. Compared to the control mice, the bone marrow of mice with loss of RBPj present a higher number of endothelial cells positive for Ki67. Additionally, we have observed that short-term specific loss of Notch signalling has leads to a dramatic increase of hematopoietic progenitor populations. Such dramatic increase can be linked to an increase proliferative status of such progenitor populations, as confirmed by Ki67/Hoescht33342 flow cytometry analyses (Fig. 6). One of the major questions that remain to be answered is whether this increase in progenitor populations does also lead to changes in their stem-like features, i.e are these progenitors able to repopulate irradiated mice in the same manner as a wild-type hematopoietic stem cell? Or does the disruption of endothelial Notch signalling result in altered “stemness” of hematopoietic stem cells? To answer this I will perform transplants of progenitor cells that I have stored frozen at -80ºC from all the experiments that I have performed and compare the ability of such cells to repopulate the irradiated bone marrow and to regenerate a new hematopoietic system. Finally, I will explore a different hematopoietic phenotype that has been consistent in all the experiments of Notch loss of function: the fact loss of Notch signaling leads to very drastic increase in the number of hematopoietic progenitors present in the peripheral blood (Figure.6C).
  • 20. Carlos LF de Castillejo Defining the endothelial-hematopoietic symbiosis 6 Figure 6. Short-term loss of Notch signalling leads to several hematopoietic phenotypes. A. Flow cytometry analyses of hematopoietic progenitor populations reveal a drastic and quite significant increase in all the progenitor populations when Notch signalling is lost. B. The increase in such populations can be attributed to a significantly higher proportion of proliferative (Ki67+) progenitor cells in mice with Notch loss of function. C. Flow cytometry analyses of peripheral blood reveal a massive increase in the amount of hematopoietic progenitors circulating in the blood in Notch loss of function mice. For all the experiments, n= 9-12 mice. From the initial application: 3. Targeting the endothelium as a possible alternative way to improve the treatment of myeloproliferative neoplasms. Unfortunately, we had to abandon this aim due to the fact that Dr. Radek Skoda did not give us his consent to use his published model of Jak2v617f neoplasms. After deliberation and consideration of the possible alternative models available, we decided that none of the available mouse models of Jak2v617f neoplasm reliably and physiologically mimicked the human neoplasms. 3(New). Could megakaryocyte progenitors act as hematopoietic stem cells and thus be able to repopulate the bone marrow and regenerate the hematopoietic system of irradiated mice?
  • 21. Carlos LF de Castillejo Defining the endothelial-hematopoietic symbiosis 7 The pursuit of this newlyproposed aim originates from initial observations and experiments in which the transplant of megakaryocyte-like cells labelled by the Venus fluorescent protein in CBF::H2B-Venus(Fig.7A).TheseCBF::H2B-VenusmiceareusedasreportermiceforNotch activity, i.e the expression of Venus denotes that the Notch signalling pathway is active in labelled cells. Interestingly, these megakaryocytic cells exhibit expression of both endothelial and hematopoietic progenitor markers (Fig.7B); which prompted us to test the possibility that these cells could share features with both of those cells types and therefore could be have the ability, as with HSCs, to rescue lethally irradiated mice. Figure 7. Under certain circumstances, megakaryocytic cells could act as hematopoietic stem cells and therefore be able to regenerate the vascular system of a lethally irradiated adult mouse. The preliminary results indicate that the transplant of Venus (+) megakaryocyte cells can indeed rescue irradiated hosts. We have monitored mice transplanted with these megakaryocyte-like cells for up to 4 months after transplant, and all of the mice in 3 different transplant experiments not only survived, but showed no vascular abnormalities and displayed robust engraftment (Fig.7C). Nevertheless, and thanks to unpublished data from the laboratory, we have also found out that the expressionof Venus might not necessarilyand accuratelyreflect the activationof the Notch signalling pathway in a cell. For this reason, we want to repeat the same type of transplant experiments that have been done with the CBF::H2B-Venus mouse line using instead another approach. We will use VE-Cadherin-CreERT2 mice intercrossed with a reporter mouse line R26-tdTomato, which will label endothelial cells and megakaryocytes (unpublished data) by their expression of the Tomato fluorescent protein. We will then isolate Tomato+ megakaryocytes and transplant them into irradiated hosts in the hopes of replicating our initial experiments withtheCBF:H2B-Venus line. If successful,we will thentest thesametransplant setting using instead VE-Cadherin-CreERT2; R26-tdTomato; RBPj lox/lox as donor mice in order to accurately describe if the repopulating capacity of megakaryocytes is dependent, and to what extent, upon the Notch signalling pathway.
  • 22. Carlos LF de Castillejo Defining the endothelial-hematopoietic symbiosis 8 Tentative work timeline For Aim 1: I will test newly generated iFUCCI mice in July 2017. If I see improvements in the expression of the Venus and Cherry fluorescent proteins I will process mice from July 2017 to September 2017 In addition, I will analyze iFUCCI mice crossed with different Notch-related alleles and repeat the same processing and quantifications performed with wild-type mice, which would be completed by March 2018. In the case that the newly generated iFUCCI mice do not meet the expectations for additional processing, I will have to abandon altogether this aim of the proposal. For Aim 2: I am performing a new set of experiments starting in August 2017 in order to collect RNA and bone samples that I can process for histology. I will perform gene expression analyses between November 2017 and January 2018. I will need to make quantifications of different proliferation markers in thin paraffin section to confirm all of my preliminary results, which will be performed between November 2017 and March 2018. For Aim 3: The mice that I need to serve as donor mice for the transplant will be generated by August 2017. I will perform the transplant in September 2017 and monitor them for at least 4 months, until the beginning of January 2018. I will then sacrifice the mice and perform the necessary histology and flow cytometry analyses, which will be achieved by February 2018. I have set-up crosses to perform similar experiments with different Notch-related alleles. As the generation of these mice require more than 2 generations, I will not be able to start this transplants until February 2018. I will then monitor the mice for at least 4 months until May 2018, and will perform the histology and flow cytometry analyses for these transplants, which will be achieved by August 2018. References Claxton, S. et al. (2008). Efficient, inducible Cre-recombinase activation in vascular endothelium. Genesis 46(2), 74-80. Kusumbe, A.P., Ramasamy, S.K., and Adams, R.H. (2014). Coupling of angiogenesis and osteogenesis by a specific vessel subtype in bone. Nature 507, 323-328. Lefrançais, E., et al.(2017) The lung is a site of platelet biogenesis and a reservoir for haemotopoetic progenitors. Nature 544, 105-109. Morrison, S.J., and Scadden, D.T. (2014). The bone marrow niche for haematopoietic stem cells. Nature 505, 327-334. Ramasamy, S.K., Kusumbe, A.P., Wang, L., and Adams, R.H. (2014). Endothelial Notch activity promotes angiogenesis and osteogenesis in bone. Nature 507, 376-380. Ramasamy, S.K., et al.(2016). Blood flow control bone vascular function and osteogenesis. Nature Communications 7, 1-13. Sorensen, I., et al.(2009). DLL1-mediated Notch activation regulates endothelial identity in mouse fetal arteries. Blood 113, 5680-5688.