Constitutively active Notch1 converts cranial neural

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© 2017. Published by The Company of Biologists Ltd | Biology Open (2017) 6, 317-325 doi:10.1242/bio.023887
RESEARCH ARTICLE
Constitutively active Notch1 converts cranial neural crest-derived
frontonasal mesenchyme to perivascular cells in vivo
ABSTRACT
Perivascular/mural cells originate from either the mesoderm or the
cranial neural crest. Regardless of their origin, Notch signalling is
necessary for their formation. Furthermore, in both chicken and mouse,
constitutive Notch1 activation (via expression of the Notch1 intracellular
domain) is sufficient in vivo to convert trunk mesoderm-derived somite
cells to perivascular cells, at the expense of skeletal muscle. In
experiments originally designed to investigate the effect of premature
Notch1 activation on the development of neural crest-derived olfactory
ensheathing glial cells (OECs), we used in ovo electroporation to insert a
tetracycline-inducible NotchΔE construct (encoding a constitutively
active mutant of mouse Notch1) into the genome of chicken cranial
neural crest cell precursors, and activated NotchΔE expression by
doxycycline injection at embryonic day 4. NotchΔE-targeted cells
formed perivascular cells within the frontonasal mesenchyme, and
expressed a perivascular marker on the olfactory nerve. Hence,
constitutively activating Notch1 is sufficient in vivo to drive not only
somite cells, but also neural crest-derived frontonasal mesenchyme and
perhaps developing OECs, to a perivascular cell fate. These results also
highlight the plasticity of neural crest-derived mesenchyme and glia.
KEY WORDS: Notch, Pericyte, Neural crest, Frontonasal
mesenchyme, Olfactory ensheathing cells, Chick embryo
Dedication
This paper is dedicated to the memory of Dr Sophie R. Miller, who passed away in
December 2016.
INTRODUCTION
Perivascular (mural) cells – pericytes and vascular smooth muscle cells –
form the periendothelial (outer) wall of blood vessels: mature pericytes
are embedded within the basement membrane of the endothelial cells in
microvessels (capillaries, terminal arterioles, postcapillary venules),
while vascular smooth muscle cells are found in multiple layers around
larger vessels (reviewed by Armulik et al., 2011; Majesky et al., 2011).
Perivascular cells in the trunk, and many in the head, originate from
mesoderm, but quail-chick chimera experiments revealed that the cranial
neural crest (including the cardiac neural crest, a subset of the cranial
neural crest that arises from the caudal hindbrain) provides perivascular
cells to blood vessels in the face, pharyngeal arches and forebrain,
including those of the retina (Le Lièvre and Le Douarin, 1975; Bergwerff
et al., 1998; Etchevers et al., 2001; Korn et al., 2002). This was later
supported via genetic lineage-tracing studies in mice (Jiang et al., 2000;
Department of Physiology, Development and Neuroscience, University of
Cambridge, Anatomy Building, Downing Street, Cambridge CB2 3DY, UK.
*Deceased
‡
Author for correspondence ([email protected])
This is an Open Access article distributed under the terms of the Creative Commons Attribution
License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,
distribution and reproduction in any medium provided that the original work is properly attributed.
Received 29 December 2016; Accepted 20 January 2017
Gage et al., 2005; Trost et al., 2013) and most recently zebrafish (Wang
et al., 2014; Ando et al., 2016).
Multiple studies over the past decade, both in vitro and in vivo, have
shown that Notch signalling is necessary for the formation of
perivascular cells originating from both the mesoderm and the neural
crest (e.g. Doi et al., 2006; Noseda et al., 2006; High et al., 2007, 2008;
Liu et al., 2009, 2010; Chang et al., 2012; Manderfield et al., 2012,
2015; Wang et al., 2014; for reviews, see Gridley, 2007, 2010; Phng and
Gerhardt, 2009; Boucher et al., 2012). Constitutive activation of the
Notch pathway, via expression of the Notch1 intracellular domain
(NICD), was sufficient to up-regulate smooth muscle myosin heavy
chain (Myh11) and other smooth muscle marker genes in the C3H10T1/
2 (mouse embryonic fibroblast) cell line (Doi et al., 2006).
Physiological Notch activation, via co-culture with L cells stably
expressing the Notch ligand Jagged1 (though not Delta-like 4), was also
sufficient to up-regulate Myh11 in this fibroblast cell line (Doi et al.,
2006). In contrast, NICD transfection did not up-regulate Myh11 in nonmesenchymal cell lines (mouse mammary gland epithelial cells, human
umbilical vein endothelial cells, or human epidermal keratinocytes)
(Doi et al., 2006). In vivo, NICD is sufficient to convert trunk
mesoderm-derived somite cells to perivascular cells, at the expense of a
muscle cell fate (Ben-Yair and Kalcheim, 2008; Mayeuf-Louchart et al.,
2014). This was first demonstrated in chicken, by electroporating the
lateral dermomyotome with NICD (Ben-Yair and Kalcheim, 2008), and
more recently in mouse, by replacing one allele of the somite-expressed
gene Pax3 with NICD (Mayeuf-Louchart et al., 2014).
Here, we show that constitutively active Notch1 is also sufficient in vivo
to drive a perivascular cell fate in cranial neural crest-derived frontonasal
mesenchyme, and perhaps also in developing olfactory ensheathing glial
cells (OECs). We originally aimed to test the effect of prematurely
activating Notch1 on the development of OECs, which are derived from
the cranial neural crest cells that colonise the frontonasal mass before the
olfactory placode forms (Barraud et al., 2010). OECs are first detected on
the chicken olfactory nerve at embryonic day (E)3.5, via immunoreactivity
for the early glial marker myelin protein zero (Mpz, P0) (Drapkin and
Silverman, 1999). Two days later, at E5.5, Notch1 is up-regulated in
developing OECs, and by E6.5, almost all developing OECs express Sox2
(Miller et al., 2016), which is a direct Notch/Rbpj target (Wakamatsu et al.,
2004; Ehm et al., 2010; Li et al., 2012). In the development of Schwann
cells, the glia of all other peripheral nerves, Notch signalling promotes the
transition from Schwann cell precursors (which express Mpz) to immature
Schwann cells (Woodhoo et al., 2009). To test the hypothesis that a similar
Notch-mediated transition is important for OEC development, we aimed
to activate Notch1 prematurely in developing chicken OECs, for which
temporal control of the onset of Notch1 signalling would be required. Sato
et al. (2008) previously used in ovo electroporation to insert into the
genome of presomitic mesoderm cells both a construct that constitutively
expresses the reverse (‘Tet-on’) tetracycline transactivator protein variant
rtTA2SM2 (Urlinger et al., 2000), and a tetracycline-inducible NotchΔE
construct, in which a single tetracycline-response element controls the
bidirectional transcription of NotchΔE (encoding a constitutively active
extracellular deletion mutant of mouse Notch1; Kopan et al., 1996) and
EGFP, whose expression was activated at somite stages by doxycycline
injection. This resulted in the conversion of somite cells either to
perivascular cells (also shown by electroporating a construct encoding
NICD directly into the lateral dermomyotome; Ben-Yair and Kalcheim,
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Sophie R. Miller*, Surangi N. Perera and Clare V. H. Baker‡
2008) or endothelial cells (Sato et al., 2008). Here, we used the conditional
expression approach of Sato et al. (2008) to insert their tetracyclineinducible NotchΔE/EGFP construct into the genome of premigratory
cranial neural crest cell precursors, and activate NotchΔE/EGFP
expression from E4 (by doxycycline injection), 1.5 days before Notch1
is normally up-regulated in developing OECs (Miller et al., 2016). To our
surprise, we saw a striking phenotype in the neural crest-derived
frontonasal mesenchyme (most of which would normally form skeletal
or connective tissue, as well as perivascular cells), namely the formation
by NotchΔE/EGFP-targeted cells of ectopic perivascular cells. NotchΔE/
EGFP-targeted cells on the olfactory nerve also upregulated a perivascular
marker. Hence, constitutive activation of Notch1 is sufficient in vivo to
convert not only trunk mesoderm-derived somite cells (Ben-Yair and
Kalcheim, 2008; Sato et al., 2008; Mayeuf-Louchart et al., 2014), but also
cranial neural crest-derived frontonasal mesenchyme (and perhaps
developing olfactory glia) to perivascular cells. These results suggest
that during normal development, vascular endothelial cells expressing
Notch ligands may recruit adjacent neural crest-derived frontonasal
mesenchyme cells (and perhaps also developing olfactory glia) to form
perivascular cells, via the sustained activation of Notch signalling.
Furthermore, given that Notch signalling was not activated in targeted
cranial neural crest-derived cells until after doxycycline was injected at E4,
several days after the end of cranial neural crest migration, our data also
speak to the plasticity of cranial neural crest-derived frontonasal
mesenchyme and developing olfactory ensheathing glia.
RESULTS
We used the Tol2 transposase/‘Tet-on’ in ovo electroporation system (Sato
et al., 2007; Watanabe et al., 2007), which inserts tetracycline-dependent
constructs into the genome of targeted cells, to drive constitutively active
Notch1 expression in cranial neural crest-derived cells from embryonic day
(E)4 [Hamburger–Hamilton (HH) stage 24; Hamburger and Hamilton,
1951]. Our original intention was to investigate the effect of premature
Notch1 activation on the development of olfactory ensheathing cells
(OECs, the glial cells of the olfactory nerve), which up-regulate Notch1
from E5.5 (HH stage 24) (Miller et al., 2016). We therefore aimed to target
the cranial neural crest precursors of OECs, which colonise the frontonasal
mass before the olfactory placode forms (Barraud et al., 2010), with the
Tol2-integratable, tetracycline-dependent construct pT2K-NotchΔE-BIEGFP (Sato et al., 2008). In this construct, a single tetracycline-response
element controls the bidirectional transcription of NotchΔE (encoding a
constitutively active extracellular deletion mutant of mouse Notch1;
Kopan et al., 1996) and EGFP (thus, EGFP labels cells successfully
targeted with NotchΔE; Sato et al., 2008).
We electroporated prospective cranial ectoderm in ovo at HH stages
6-8 (25-28 h of incubation) with pT2K-NotchΔE-BI-EGFP (hereafter
NotchΔE/EGFP) or the Tol2-integratable control construct pT2KCAGGS-EGFP, encoding EGFP only (Sato et al., 2007). Each of these
constructs was co-electroporated with the Tol2-integratable construct
pT2K-CAGGS-rtTA2SM2 (Sato et al., 2007), encoding the reverse (‘Teton’) tetracycline transactivator protein rtTA2 under the control of the
synthetic CAGGS promoter (Niwa et al., 1991) (thus providing a
continuous supply of rtTA2 in targeted cells), plus the pCAGGS-T2TP
construct, encoding Tol2 transposase (Sato et al., 2007) (to insert the
rtTA2 and NotchΔE/EGFP or control EGFP constructs into the genome
of targeted cells). Doxycycline was injected into the yolk under the
embryo at E4 (HH stage 24) to initiate NotchΔE/EGFP expression (the
control EGFP is constitutively expressed). Embryos were collected
1-4 days later (E5-E8; HH stages 27-34) for sectioning, followed by
in situ hybridisation plus immunohistochemistry on sections.
Constitutive Notch activation from E4 converts frontonasal
mesenchyme cells to perivascular cells
At E6 (HH stage 29; two days after doxycycline injection) in control
EGFP-targeted embryos (n=2), EGFP-positive cells are distributed
throughout the frontonasal mesenchyme and along peripheral nerves
(Fig. 1A-B1), with only a few EGFP-positive cells associated with Lmo2positive vascular endothelium (Nagai and Sheng, 2008) (Fig. 1C-D2). In
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Biology Open (2017) 6, 317-325 doi:10.1242/bio.023887
contrast, in NotchΔE/EGFP-targeted embryos at E6-7 (HH stages 29-31;
n=8), most EGFP-positive cells are aggregated in rings in the
mesenchyme (Fig. 1E-F1), encircling Lmo2-positive vascular
endothelium (Fig. 1G-H2). The same ‘ring-like’ distribution of EGFPpositive cells was also seen in NotchΔE/EGFP-targeted embryos at E5
(HH stage 27; n=3) (not shown). Notch pathway activation in NotchΔE/
EGFP-targeted cells at E6 was confirmed by co-immunostaining for
EGFP and the cleaved Notch1 intracellular domain (n=2; Fig. 1I-J1).
Since cranial neural crest cells normally give rise to perivascular cells
in the blood vessels of the face and forebrain (Etchevers et al., 2001), we
wished to use molecular markers to test whether the NotchΔE/EGFPtargeted cells encircling Lmo2-positive vascular endothelium in the
frontonasal mesenchyme were indeed adopting a perivascular cell fate.
There are no exclusive molecular markers for perivascular cells;
furthermore, the expression levels of the various markers used can vary,
depending on, for example, the developmental state of the cells
(reviewed by Armulik et al., 2011). Nevertheless, one commonly used
perivascular cell marker is platelet-derived growth factor receptor beta
(Pdgfrb) (reviewed by Armulik et al., 2011). After doxycycline
injection at E4, control EGFP-targeted embryos at E6 show almost no
co-localisation between EGFP and Pdgfrb (n=3), barring a few cells
associated with the vasculature, as expected (Fig. 2A-B2). In contrast,
most NotchΔE/EGFP-targeted cells in the frontonasal mesenchyme
express Pdgfrb at E6-7 (n=5) (Fig. 2C-D2). Perivascular cells also
express vascular endothelial growth factor A (Vegfa) (Darland et al.,
2003; Parenti et al., 2004; Kale et al., 2005). After initiating constitutive
Notch activity by injecting doxycycline at E4, we detected Vegfa
expression in NotchΔE/EGFP-targeted cells at E6-E7 (n=2; Fig. 2G-H2).
Furthermore, immunoreactivity for the smooth muscle/myofibroblast
marker alpha-smooth muscle actin (Acta2; reviewed by Armulik et al.,
2011) was detected in some NotchΔE/EGFP-targeted cells associated
with larger blood vessels at E5-E8 (n=2; Fig. 2I-J2).
Overall, these data suggest that constitutive Notch activation from E4
in cranial neural crest-derived frontonasal mesenchyme cells is
sufficient to convert them to perivascular cells, identified by their
location (i.e. encircling vascular endothelial cells in developing blood
vessels) in combination with the expression of characteristic
perivascular cell markers.
Constitutive Notch activation from E4 may convert developing
olfactory ensheathing cells into perivascular cells
In control EGFP-targeted embryos at E6 (two days after doxycycline
injection), EGFP-positive developing OECs (which are neural crestderived; Barraud et al., 2010) are distributed throughout the olfactory
nerve, among the axons (Fig. 3A-B1). In contrast, in NotchΔE/EGFPtargeted embryos at E6 (n=4), most NotchΔE/EGFP-targeted cells on the
olfactory nerve seem to be excluded from the nerve’s interior, instead
aggregating at the edges of the nerve in ‘processes’ extending away from it
(Fig. 3C-D1). At least some NotchΔE/EGFP-targeted cells on the olfactory
nerve at E6-7 express the perivascular cell marker Pdgfrb (n=2; Fig. 3E-E2),
suggesting that, like NotchΔE/EGFP-targeted cells in the frontonasal
mesenchyme, they may have been converted to perivascular cells. Several
of the NotchΔE/EGFP-targeted cells on the olfactory nerve express the
OEC marker Sox10 (Barraud et al., 2010) (Fig. 3E-E2), confirming that at
least some developing OECs were targeted. Indeed, a few of the NotchΔE/
EGFP-targeted cells co-express Sox10 and Pdgfrb (yellow arrowheads,
Fig. 3E-E2), suggesting they may have been caught in the process of
changing fate. Some of the NotchΔE/EGFP-targeted cells on the olfactory
nerve are Pdgfrb-positive but Sox10-negative (black/white arrowheads,
Fig. 3E-E2): these may have originated from NotchΔE/EGFP-targeted
developing OECs that have already down-regulated Sox10 expression, or
NotchΔE/EGFP-targeted frontonasal mesenchyme cells that have
colonised the nerve. The endogenous olfactory nerve microvasculature
is starting to form at this time; in situ hybridisation for Pdgfrb and the
vascular endothelial cell marker Lmo2 on sections of both NotchΔE/
EGFP-targeted and wild-type embryos at E6.5-7 (n=3) reveals some
untargeted Pdgfrb-positive cells (red arrowheads, Fig. 3E-E2) and a few
Lmo2-positive cells (Fig. 3F-F2) within the olfactory nerve.
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RESEARCH ARTICLE
Biology Open (2017) 6, 317-325 doi:10.1242/bio.023887
Vasculature containing NotchΔE/EGFP-targeted perivascular
cells seems to attract peripheral axons and glia
In half of the NotchΔE/EGFP-targeted embryos at E5-8 (n=6 out of
12), olfactory and other peripheral axons and their accompanying
OECs/Schwann cells seemed to project towards vasculature
containing NotchΔE/EGFP-targeted cells, with some of the glial
cells (identified by Sox10 expression; Barraud et al., 2010; Jacob,
2015) even found isolated from axons, in association with such cells.
Fig. 4A-A3 shows an example at E5, in which the olfactory nerve is in
contact with such a blood vessel, at which point olfactory axons seem
to project in the wrong direction, away from the forebrain. Fig. 4B-B3
shows an example at E6, in which the olfactory nerve is in close
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Fig. 1. Constitutive Notch activation from E4 causes frontonasal mesenchyme cells to associate with vascular endothelial cells. Parasagittal sections of the
olfactory region from chicken embryos in which the cranial ectoderm had been targeted in ovo at E1 with EGFP alone (control) or NotchΔE/EGFP, using the Tol2
transposase/‘Tet-on’ electroporation system (Sato et al., 2007; Watanabe et al., 2007). Eggs were injected with doxycycline at E4. (A-B1) A control EGFP-targeted
embryo at E6. Immunostaining for EGFP and Tubb3 reveals EGFP-targeted cells throughout the mesenchyme and along the olfactory nerve, as well as in the
forebrain, surface ectoderm and olfactory epithelium. (C-D2) In the same embryo at E6, EGFP-targeted cells are uniformly distributed in the frontonasal
mesenchyme; only a few are associated with Lmo2-positive vascular endothelial cells (arrowheads in D-D2 highlight examples). (E-F1) A NotchΔE/EGFP-targeted
embryo at E6. NotchΔE/EGFP-targeted cells aggregate in rings in the frontonasal mesenchyme (arrowheads in F,F1) and at the edges of the olfactory nerve. (G-H2)
In the same embryo at E6, NotchΔE/EGFP-targeted cells encircle Lmo2-positive vascular endothelial cells. (I) The same NotchΔE/EGFP-targeted embryo at E6
shown in E-F1. Immunostaining for EGFP and Tubb3 shows rings of NotchΔE/EGFP-targeted cells in the frontonasal mesenchyme and along the edges of olfactory
nerve. (J,J1) Higher-power view of the boxed region in I. Immunostaining for the cleaved Notch1 intracellular domain reveals co-localisation with NotchΔE/EGFPtargeted cells (arrowheads highlight examples, both in the mesenchyme and at the edges of the olfactory nerve). bv, blood vessel; EGFP, enhanced GFP; fb,
forebrain; NICD, cleaved Notch1 intracellular domain; oe, olfactory epithelium; on, olfactory nerve. Scale bars: 100 µm.
RESEARCH ARTICLE
Biology Open (2017) 6, 317-325 doi:10.1242/bio.023887
Fig. 2. Constitutive Notch activation from E4 converts frontonasal mesenchyme cells to perivascular cells. Parasagittal sections of the frontonasal region
from chicken embryos in which the cranial ectoderm had been targeted in ovo at E1 with EGFP alone (control) or NotchΔE/EGFP, using the Tol2 transposase/‘Tet-on’
electroporation system. Eggs were injected with doxycycline at E4. (A) A control EGFP-targeted embryo at E6. In situ hybridisation for Pdgfrb reveals perivascular
cells in rings in the frontonasal mesenchyme and at the edge of the forebrain. (B) Higher-power view of boxed region in A, showing Pdgfrb-positive perivascular cells
associated with a blood vessel near the forebrain. (B1,B2) Same section as B immunostained for EGFP, with Pdgfrb shown as a false-colour overlay in B2, reveals a
fairly uniform distribution of EGFP-targeted cells in the mesenchyme and almost no co-localisation with Pdgfrb, apart from a few cells (arrowheads). (C-D2) A
NotchΔE/EGFP-targeted embryo at E6. NotchΔE/EGFP-targeted cells are found in rings around the developing blood vessels and express Pdgfrb (arrowheads
highlight examples), showing they are perivascular cells. (E-F2) A NotchΔE/EGFP-targeted embryo at E7. Blood vessels in the frontonasal mesenchyme (arrows
indicate blood cells) are surrounded by NotchΔE/EGFP-targeted, Pdgfrb-positive perivascular cells (arrowheads). (G) In the same NotchΔE/EGFP-targeted embryo
at E7, Vegfa is expressed in developing vasculature in the frontonasal mass. (H-H2) Higher-power view of the boxed region in G, revealing Vegfa-positive NotchΔE/
EGFP-targeted cells (arrowheads). (I-J2) A NotchΔE/EGFP-targeted embryo at E5, with NotchΔE/EGFP-targeted cells in rings in the frontonasal mesenchyme.
Immunostaining for alpha-smooth muscle actin (Acta2) reveals a few Acta2-positive NotchΔE/EGFP-targeted cells (arrowheads) associated with a large blood
vessel near the olfactory nerve. bv, blood vessel; EGFP, enhanced GFP; fb, forebrain; on, olfactory nerve. Scale bars: 100 µm.
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containing NotchΔE/EGFP-targeted cells (Fig. 4I-I3). Taken together,
these results suggest that vasculature containing NotchΔE/EGFPtargeted cells attracts both peripheral axons and glia.
DISCUSSION
In experiments originally aimed at testing the effect on olfactory
ensheathing cell (OEC) development of prematurely activating Notch1,
which is normally expressed in developing chicken OECs from E5
(Miller et al., 2016), we used the Tol2 transposase/‘Tet-on’ in ovo
electroporation system (Sato et al., 2007; Watanabe et al., 2007) to drive
NotchΔE, encoding a constitutively active form of mouse Notch1
(Kopan et al., 1996; Sato et al., 2008), in cranial neural crest-derived
cells from E4. This proved to be sufficient to convert both frontonasal
mesenchyme cells, and perhaps also developing OECs, to Pdgfrb-
Biology Open
contact with vasculature containing NotchΔE/EGFP-targeted cells,
towards which untargeted Sox10-positive OECs seem to have
migrated, leaving the olfactory nerve altogether. Fig. 4C-G2 shows
another example at E7, in which NotchΔE/EGFP-targeted, Pdgfrbpositive perivascular cells are closely associated with Sox10-positive
glial cells (Fig. 4D1,D2) and axons (and possibly neurons) caudal to
the olfactory system (Fig. 4G1,G2). In one NotchΔE/EGFP-targeted
embryo at E8 (HH stage 34), the entire olfactory nerve on one side is
misplaced laterally, outside the cartilage that normally encloses it,
apparently projecting towards large blood vessels containing
NotchΔE/EGFP-targeted cells (Fig. 4H). On a nearby section from
the same embryo, many Sox10-positive OECs (both NotchΔE/EGFPtargeted and untargeted) seem to have migrated away from the
olfactory nerve altogether, instead associating with blood vessels
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Biology Open (2017) 6, 317-325 doi:10.1242/bio.023887
positive perivascular cells. Pdgfrb encodes a receptor tyrosine kinase
required in pericytes during angiogenesis, for their recruitment to
sprouting capillaries and proliferation (Lindahl et al., 1997; Hellström
et al., 1999; Winkler et al., 2010). In the frontonasal mesenchyme at
E5-7, ectopic NotchΔE/EGFP-targeted perivascular cells were found
encircling Lmo2-positive vascular endothelium. Vegfr2 (Flk1, Kdr)expressing angioblasts are found throughout the developing cranial
mesenchyme in both chicken and mouse (Couly et al., 1995; Yoshida
et al., 2008); in chicken, these initially dispersed Vegfr2-positive cells
have all incorporated into blood vessels by E3-4 (Couly et al., 1995).
Hence, expression of constitutively active Notch1 from E4 in cranial
neural crest-derived frontonasal mesenchyme cells causes them to adopt
a perivascular cell fate and associate with the vascular endothelium of
nearby blood vessels.
NotchΔE/EGFP-targeted Pdgfrb-positive cells were also seen within
the olfactory nerve, suggesting that constitutive Notch1 activation from
E4 within developing OECs (which can first be identified at E3.5, by
myelin protein zero immunoreactivity; Drapkin and Silverman, 1999)
could be sufficient to convert them to a perivascular cell fate. Indeed,
some of the NotchΔE/EGFP-targeted, Pdgfrb-positive cells on the
olfactory nerve co-expressed the OEC marker Sox10 (Barraud et al.,
2010), suggesting they were in the process of changing fate. Most
NotchΔE/EGFP-targeted cells seemed to be excluded from the interior
of the olfactory nerve and instead aggregated together at the edges,
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Fig. 3. Constitutive Notch activation from E4 converts developing olfactory ensheathing cells into perivascular cells. Parasagittal sections of the olfactory
region from chicken embryos in which the cranial ectoderm had been targeted in ovo at E1 with EGFP alone (control) or NotchΔE/EGFP, using the Tol2 transposase/
‘Tet-on’ electroporation system. Eggs were injected with doxycycline at E4. Dotted lines demarcate the olfactory nerve. (A-B1) A control EGFP-targeted embryo at E6,
in which the olfactory placode was not targeted. EGFP-targeted neural crest-derived cells are found throughout the frontonasal mesenchyme and associated with
Tubb3-positive olfactory axons, presumably developing OECs. (C-D1) A NotchΔE/EGFP-targeted embryo at E6. NotchΔE/EGFP-targeted cells associated with the
olfactory nerve are aggregated at the edges of the nerve, rather than being found throughout the nerve, and form processes extending away from it (arrowhead in D,
D1). (E-E2) In a NotchΔE/EGFP-targeted embryo at E7, in situ hybridisation for the perivascular marker Pdgfrb (shown as a false-colour overlay in E1,E2), combined
with immunostaining for the OEC marker Sox10, shows that some NotchΔE/EGFP-targeted cells are developing OECs; a few of these co-express Pdgfrb (yellow
arrowheads), suggesting they may be undergoing fate conversion. Some NotchΔE/EGFP-targeted cells express Pdgfrb but not Sox10 (black/white arrowheads).
Some untargeted cells express Pdgfrb (red arrowheads). (F-F2) In a nearby section from the same NotchΔE/EGFP-targeted embryo at E7, in situ hybridisation for
Lmo2 reveals a few weakly Lmo2-positive vascular endothelial cells on the olfactory nerve (arrowheads). bv, blood vessel; EGFP, enhanced GFP; fb, forebrain; oe,
olfactory epithelium; on, olfactory nerve; pn, peripheral nerve. Scale bars: 100 μm.
Biology Open (2017) 6, 317-325 doi:10.1242/bio.023887
Fig. 4. Peripheral axons and glia seem to be attracted to blood vessels containing NotchΔE/EGFP-targeted cells. Parasagittal (A-G2) and coronal (H-I3)
sections from embryos in which the cranial ectoderm had been targeted in ovo at E1 with NotchΔE/EGFP, using the Tol2 transposase/‘Tet-on’ electroporation
system. Eggs were injected with doxycycline at E4. (A) In an E5 embryo, in situ hybridisation for Sox10 reveals developing OECs on the olfactory nerve.
(A1-A3) Same section as A, immunostained for EGFP and Tubb3, with Sox10 shown as a false-colour overlay in A2,A3. A thin nerve branch (arrow) deviates from
the olfactory nerve away from the forebrain (for orientation, see low-power inset in A2). The branch-point is near a developing blood vessel, whose wall contains
NotchΔE/EGFP-targeted cells. (B-B3) In an E6 embryo, several untargeted Sox10-positive cells (arrowheads), presumably developing OECs, are found isolated
in the mesenchyme at some distance from the olfactory nerve, near NotchΔE/EGFP-targeted cells. (C-D2) In an E7 embryo, in situ hybridisation for Pdgfrb
followed by immunostaining for EGFP and Sox10 reveals that many NotchΔE/EGFP-targeted cells have formed Pdgfrb-positive perivascular cells, with which
many Sox10-positive cells ( presumably peripheral glial cells) are associated. This is far from the olfactory nerve: note the location of the olfactory epithelium at the
top right. (E-G2) A nearby section of the same E7 embryo, shown at low-power in E-E3 for orientation (note the position of the olfactory epithelium and olfactory
nerve towards the top right, and the forebrain and adenohypophysis towards the top left). In situ hybridisation for Lmo2 and immunostaining for EGFP and Tubb3
confirm the presence of peripheral axons (and possibly neurons) close to a large concentration of NotchΔE/EGFP-targeted cells that are associated with
Lmo2-positive vascular endothelium. (H) In an E8 embryo (coronal section), the entire olfactory nerve on one side is misplaced laterally (yellow arrow) towards
several large blood vessels whose walls contain NotchΔE/EGFP-targeted cells. The displaced olfactory nerve is in contact with another peripheral nerve, and no
longer surrounded by cartilage (identified by immunostaining with an anti-Sox9 antibody that also cross-reacts with other SoxE family members), unlike the
olfactory nerve on the other side. (I-I3) In a nearby section of the same E8 embryo, in situ hybridisation for Sox10 and immunostaining for EGFP and Tubb3 show
that some Sox10-positive OECs – both untargeted (black/white arrowheads) and NotchΔE/EGFP-targeted (yellow arrowheads) – are found at a distance from
axons, associated instead with blood vessels whose walls contain NotchΔE/EGFP-targeted cells. ah, adenohypophysis; bv, blood vessel; EGFP, enhanced GFP;
fb, forebrain; oe, olfactory epithelium; on, olfactory nerve; pn, peripheral nerve. Scale bars: 100 µm.
projecting away from the nerve. This may reflect the lack of blood
vessels inside developing nerves until relatively late in development,
given that we did not see many Lmo2-positive vascular endothelial cells
inside the chicken olfactory nerve at E6.5-7 (in the rat sciatic nerve,
322
blood vessels are first seen only at E18; Wanner et al., 2006). The
presence of some untargeted Pdgfrb-positive cells within the olfactory
nerve at E6.5-7 also suggests that perivascular cells are normally
beginning to differentiate at this stage. Taken together, these data may
Biology Open
RESEARCH ARTICLE
RESEARCH ARTICLE
cells attracts OECs/Schwann cells, and at least in some cases olfactory
axons, towards the vasculature.
Overall, our data support and extend previous work showing that the
Notch pathway is necessary for the formation of perivascular cells from
the cranial neural crest (High et al., 2007, 2008; Chang et al., 2012;
Manderfield et al., 2012; Wang et al., 2014; Manderfield et al., 2015),
by showing that constitutively active Notch1 promotes a perivascular
cell fate in frontonasal mesenchyme, and perhaps also in glial
progenitors on the olfactory nerve, several days after the end of
cranial neural crest migration. Intriguingly, constitutive activation of
Notch signalling via expression of the Notch3 intracellular domain
seems to promote the proliferation, but not the specification, of brain
pericytes in zebrafish (Wang et al., 2014), suggesting that the activation
of distinct Notch signalling pathways may have different outcomes
during the development of perivascular cells.
MATERIALS AND METHODS
Electroporation constructs
All electroporation constructs were kind gifts of Yoshiko Takahashi (Kyoto
University, Kyoto, Japan); the pT2K-NotchΔE-BI-EGFP construct (Sato
et al., 2008) was used with the kind permission of Raphael Kopan
(Washington University, St Louis, MO, USA). Constructs were prepared
using the EndoFree Plasmid Maxi kit (Qiagen) to a stock concentration of
5 μg/μl. pCAGGS-T2TP (Kawakami and Noda, 2004; Sato et al., 2007)
encodes Tol2 transposase under the control of the synthetic CAGGS
promoter (Niwa et al., 1991); the Tol2-integratable pT2K-CAGGSrtTA2SM2 construct (Sato et al., 2007) encodes the reverse (‘Tet-on’)
tetracycline transactivator protein variant rtTA2SM2 (Urlinger et al., 2000);
the Tol2-integratable, tetracycline-dependent pT2K-NotchΔE-BI-EGFP
construct (Sato et al., 2008) encodes a constitutively active extracellular
deletion mutant of mouse Notch1 (NotchΔE; Kopan et al., 1996) and EGFP,
bidirectionally transcribed under the control of a single tetracyclineresponse element; the Tol2-integratable pT2K-CAGGS-EGFP control
construct (Sato et al., 2007) encodes EGFP alone.
In ovo electroporation
Fertilised chicken (Gallus gallus domesticus) eggs were obtained from
commercial sources. All work with chicken embryos was conducted in
accordance with the UK Animals (Scientific Procedures) Act 1986. Eggs were
incubated in a humidified atmosphere at 38°C for 25-28 h to reach
Hamburger–Hamilton stages 6-8 (Hamburger and Hamilton, 1951)
(between the head-fold stage and the 4-somite stage). Black ink (Fount
India, Pelikan) was diluted to 1% in filtered phosphate-buffered saline (PBS)
and injected underneath the blastoderm to visualise the embryo. The cranial
ectoderm and neural folds were co-electroporated with 1:1:1 pCAGGS-T2TP,
pT2K-CAGGS-rtTA2SM2 and either pT2K-NotchΔE-BI-EGFP or control
pT2K-CAGGS-EGFP, to a final concentration of 0.9 μg/μl each, mixed with
Fast Green to a final dilution of 2% and sucrose to a final concentration of 8%.
The positive electrode was placed in the yolk underneath the head process and
perpendicular to the cranial–caudal axis of the embryo. The plasmid solution
was micro-pipetted over the cranial ectoderm and the negative ‘spoon-type’
electrode brought down over the embryo, as described (Brown et al., 2012).
An ECM 830 Square Wave Pulse generator (BTX Instrument Division,
Harvard Apparatus, Inc.) was used to apply five 50-ms 5 V pulses at 100 ms
intervals. The egg was sealed with Parafilm and returned to the incubator. At
embryonic day (E)4, 500 μl of doxycycline solution (100 μg/μl doxycycline
in water) was injected under the embryo. The egg was re-sealed and returned
to the incubator until the desired stage. Surviving embryos were fixed in
modified Carnoy’s (6 volumes ethanol, 3 volumes 37% formaldehyde, 1
volume glacial acetic acid), dehydrated into ethanol, cleared in Histosol
(National Diagnostics) and embedded in paraffin wax for sectioning at 6 μm
on a rotary microtome (Microm).
Riboprobes
Chicken Lmo2 (Nakazawa et al., 2006) was a kind gift of Guojun Sheng
(RIKEN Center for Developmental Biology, Kobe, Japan). Chicken Sox10
(Cheng et al., 2000) was a kind gift of Marianne Bronner (Caltech,
Pasadena, CA, USA). An 803-bp fragment of chicken Pdgfrb cDNA,
323
Biology Open
also suggest that at least some of the perivascular cells of the olfactory
nerve vasculature derive from developing OECs, in response to
sustained Notch1 activation. This is in contrast to the trunk, where
only endoneurial fibroblasts, and not endoneurial perivascular cells,
derive from Schwann cell precursors (Joseph et al., 2004). Furthermore,
since expression of the constitutively active Notch1 mutant protein was
only activated in targeted cranial neural crest-derived cells following
doxycycline injection at E4, our findings also reveal the plasticity of
cranial neural crest-derived frontonasal mesenchyme and developing
olfactory ensheathing glia.
Our results are consistent with previous work showing that
constitutive Notch1 activation (via expression of the Notch1
intracellular domain) in trunk mesoderm-derived somite cells
promotes adoption of a perivascular fate at the expense of a skeletal
muscle fate (Ben-Yair and Kalcheim, 2008; Sato et al., 2008; MayeufLouchart et al., 2014); they also extend this finding to cranial neural
crest-derived cells. The Notch pathway plays critical roles in many
aspects of vascular development, including perivascular cell
recruitment and differentiation during vasculogenesis (i.e. the
formation of new blood vessels de novo) in addition to maturation,
stabilization and remodelling of the vasculature during angiogenesis
(i.e. the formation of new blood vessels by sprouting from existing
vessels) (reviewed by Gridley, 2007; Phng and Gerhardt, 2009; Gridley,
2010; Boucher et al., 2012). Our data suggest that constitutive Notch1
signalling from E4 in cranial neural crest-derived frontonasal
mesenchyme and developing OECs promotes a perivascular cell fate.
Since Notch signalling is required for neural crest-derived perivascular
cell formation (High et al., 2007, 2008; Chang et al., 2012; Manderfield
et al., 2012; Wang et al., 2014; Manderfield et al., 2015), this likely
reflects a normal developmental process, whereby vascular endothelial
cells expressing Notch ligands recruit adjacent frontonasal mesenchyme
cells to form perivascular cells through sustained activation of Notch
signalling.
Consistent with this hypothesis, sustained activation of Notch
signalling [via exposure to Delta-like 4 (Dll4) from endothelial cells]
is both sufficient and necessary for conversion of skeletal myoblasts to
pericytes in vitro: silencing of Dll4 restores myogenesis (Cappellari
et al., 2013). In vivo, expression of the Notch1 intracellular domain in
MyoD-positive muscle cells also drives a pericyte fate, while occasional
perivascular cells in wild-type embryos are derived from Myf5- or
MyoD-expressing precursors (Cappellari et al., 2013). This suggests
that Notch ligand production from vascular endothelium in skeletal
muscle may sometimes induce a fate switch in adjacent myoblasts.
Sustained Notch signalling is also required in vascular smooth muscle
cells to suppress alternative fates and maintain the perivascular fate: in
the absence of the Notch ligand Jagged1, mouse somite-derived
vascular smooth muscle cells adopt a chondrocyte fate, which can lead
to vessel ossification (Briot et al., 2014). Thus, sustained Notch
signalling appears not only to promote, but also maintain, the
perivascular cell fate.
We also found that vasculature containing NotchΔE/EGFP-targeted
perivascular cells seemed to attract peripheral axons and their associated
glia (OECs on the olfactory nerve; Schwann cells on all other nerves), with
some Sox10-positive glial cells appearing to have left the nerve altogether.
We identified Vegfa expression in NotchΔE/EGFP-targeted perivascular
cells. Vegfa is expressed by pericytes in the developing retinal vasculature
(where pericytes are neural crest-derived; Etchevers et al., 2001; Trost
et al., 2013); in heterozygous VegfalacZ transgenic mice (in which lacZ
under an independent ribosome entry site was inserted into the 3′
untranslated region of the Vegfa gene; Miquerol et al., 1999), retinal
pericytes express beta-galactosidase (Darland et al., 2003). Vegfa is also
secreted by perivascular cells induced from 10T1/2 cells by co-culturing
with endothelial cells (Darland et al., 2003). Vegfa is not only a proangiogenic factor (reviewed by Jin et al., 2014; Moens et al., 2014) but is
also secreted by Schwann cells, acting in an autocrine loop to enhance
Schwann cell proliferation and migration, and also promoting axon
outgrowth via Vegfr2 (reviewed by Rosenstein et al., 2010). Thus it is
possible that Vegfa secreted by NotchΔE/EGFP-targeted perivascular
Biology Open (2017) 6, 317-325 doi:10.1242/bio.023887
RESEARCH ARTICLE
In situ hybridisation on sections
Slides were de-waxed in Histosol (National Diagnostics) and rehydrated
through a graded ethanol series into diethylpyrocarbonate (DEPC)-treated
PBS. Digoxigenin-labelled antisense riboprobes diluted 1:250 to 1:1000 in
hybridisation buffer [1x salt solution (0.2 M NaCl, 10 mM Tris pH 7.5,
5 mM NaH2PO4.H2O, 5 mM Na2HPO4, 5 mM EDTA), 50% formamide,
10% dextran sulfate, 1 mg/ml yeast tRNA, 1x Denhardt’s solution] were
hybridised to sections overnight at 68°C. Slides were washed three times in
wash solution (50% formamide, 1× SSC, 0.1% Tween-20) for 30 min to one
hour each at 70°C, then given two 10-min washes in MABT (1x maleic acid
buffer with 0.1% Tween-20) (10× MAB: 1 M maleic acid, 1.5 M NaCl, pH
7.5) at room temperature. Slides were incubated for at least 2 h in blocking
solution [1% blocking reagent (Roche), 20% heat-denatured normal
sheep serum (Sigma) in MABT]. Alkaline phosphatase-conjugated antidigoxigenin antibody (Roche) was diluted 1:1500 in blocking solution
and slides were incubated in the antibody solution overnight at room
temperature. After five 30-min washes in MABT, slides were equilibrated
via two 10-min washes in NTMT (0.1 M NaCl, 0.1 M Tris, pH 9.5, 50 mM
MgCl2, 0.1% Tween-20), and the colour reaction performed in 20 μl/ml
NBT/BCIP (Roche) in NTMT. Once the colour had developed to the desired
extent, sections were washed twice in distilled water and once in PBS, then
fixed for 5 min in 4% formaldehyde (Thermo Scientific) in PBS.
Immunohistochemistry
Whether after fixation following in situ hybridisation as described in the
preceding section, or after de-waxing and rehydrating untreated slides as
described in the preceding section, slides were rinsed in PBS, blocked for 1 h
at room temperature in 10% sheep serum in PBS with 0.1% Triton X-100 and
then incubated overnight at 4°C with primary antibodies in blocking solution.
(When the antibody against cleaved Notch1 intracellular domain was used,
antigen retrieval was performed prior to blocking, by heating the slides for
4 min until boiling in a microwave in 10 mM sodium citrate buffer solution,
pH 6, followed by two washes in PBS.) After three 5-10 min washes in PBS,
appropriately matched Alexa Fluor-conjugated secondary antibodies
(Molecular Probes) were applied at 1:1000 in the same blocking solution
and incubated at room temperature for 2-3 h. If three primary antibodies were
used, a biotinylated (instead of Alexa Fluor-conjugated) secondary antibody
was used against Tubb3 (1:50 goat anti-mouse IgG2a, Invitrogen, or 1:250
horse anti-mouse IgG, Vector Laboratories), and, after three 5-10 min washes
in PBS, the slides were further incubated for 1-2 h at room temperature with
Alexa Fluor 350-conjugated NeutrAvidin (Molecular Probes) diluted 1:100 in
filtered PBS. After three 5-10 min washes in PBS, slides were mounted in
Fluoromount G (Southern Biotech). Primary antibodies used were: anti-Acta2
(mouse IgG2a, Sigma-Aldrich A5228, 1:500); anti-EGFP (rabbit, Invitrogen
A-6455, 1:500; mouse IgG1, Roche 1814460001, 1:500); anti-activated
Notch1 (cleaved Notch1 intracellular domain) (rabbit, Abcam ab8925,
1:150); anti-Sox10 (Meng et al., 2011; Yardley and García-Castro, 2012)
(rabbit, kind gift of Vivian Lee, Medical College of Wisconsin, WI, USA,
1:3000); anti-Tubb3 (neuronal class III beta-tubulin) (clone TUJ1, mouse
IgG2a, Covance MMS-435P, 1:500).
324
Image capture and processing
Images were captured on a Zeiss AxioSkop 2 MOT compound microscope
using QCapture Pro 6.0 software, a QImaging Retiga 2000R camera and an
RGB pancake (QImaging), and processed using Adobe Photoshop CS6. To
show co-localisation, bright-field in situ hybridisation images were inverted
and inserted into the green channel only, then used as a false-colour overlay
with immunofluorescence images of the same section.
Acknowledgements
Thanks to Yoshiko Takahashi (Kyoto University, Kyoto, Japan), Raphael Kopan
(Washington University, St Louis, MO, USA), Marianne Bronner (Caltech,
Pasadena, CA, USA) and Guojun Sheng (RIKEN Center for Developmental Biology,
Kobe, Japan) for providing plasmids. Thanks to Vivian Lee (Medical College of
Wisconsin, Milwaukee, WI, USA) for providing the anti-Sox10 antibody.
Competing interests
The authors declare no competing or financial interests.
Author contributions
C.V.H.B. and S.R.M. designed the study and wrote the paper. S.R.M. performed all
of the experiments, analysed the data and prepared the figures. S.N.P. contributed
some in situ hybridisation and immunostaining data used in the analysis.
Funding
S.R.M. was supported by a PhD research studentship from the Anatomical Society,
with additional funding from the Cambridge Philosophical Society. S.N.P. was
supported by the Wellcome Trust (PhD Studentship 102453/Z/13/Z) and the
Cambridge Commonwealth Trust.
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