© 2015. Published by The Company of Biologists Ltd | Development (2015) 142, 1193-1202 doi:10.1242/dev.119529
RESEARCH ARTICLE
TECHNIQUES AND RESOURCES
Second-generation Notch1 activity-trap mouse line (N1IP::CreHI)
provides a more comprehensive map of cells experiencing Notch1
activity
ABSTRACT
We have previously described the creation and analysis of a Notch1
activity-trap mouse line, Notch1 intramembrane proteolysis-Cre6MT or
N1IP::CreLO, that marked cells experiencing relatively high levels of
Notch1 activation. Here, we report and characterize a second line with
improved sensitivity (N1IP::CreHI) to mark cells experiencing lower levels
of Notch1 activation. This improvement was achieved by increasing
transcript stability and by restoring the native carboxy terminus of Cre,
resulting in a five- to tenfold increase in Cre activity. The magnitude of this
effect probably impacts Cre activity in strains with carboxy-terminal Ert2
fusion. These two trap lines and the related line N1IP::CreERT2 form a
complementary mapping tool kit to identify changes in Notch1 activation
patterns in vivo as the consequence of genetic or pharmaceutical
intervention, and illustrate the variation in Notch1 signal strength from one
tissue to the next and across developmental time.
KEY WORDS: Activity, Cre, Neuron, Notch1, Kidney
INTRODUCTION
The Notch signaling pathway is an evolutionarily conserved, shortrange communication mechanism utilized throughout life in all
metazoan species (Artavanis-Tsakonas et al., 1999). The binding of a
ligand to a Notch receptor triggers the unfolding of a juxtamembrane
negative regulatory region (NRR), which exposes it to cleavage by the
ADAM10 protease (a disintegrin and metalloprotease) at an
extracellular sequence termed S2 (Groot et al., 2013; Mumm et al.,
2000; van Tetering et al., 2009). S2 cleavage leads to the shedding of
the extracellular domain and allows γ-secretase to cleave Notch within
its transmembrane domain at S3, releasing the intracellular domain of
Notch (NICD) from the membrane. Three nuclear localization signals
within NICD provide for nuclear translocation, where it complexes
with RBPjk, recruits the MAML adaptor and the transcriptional
activator p300 (Ep300 – Mouse Genome Informatics) to the
promoters of target genes (Kopan and Ilagan, 2009).
Whereas only one receptor (Notch) and two ligands (Delta and
Serrate) are present in Drosophila, mammals have four receptor
paralogs (Notch1-4) and at least five canonical ligands (Dll1, Dll3,
Dll4, Jag1 and Jag2). These receptors and ligands exhibit complex
expression patterns and play essential roles both during
development and in the maintenance of adult tissue homeostasis,
1
2
SAGE Labs, St Louis, MO 63146, USA. Department of Developmental Biology,
3
Washington University, St Louis, MO 63110, USA. Division of Developmental
4
Biology, Children’s Hospital Medical Center, Cincinnati, OH 45229, USA. Carnegie
Institution for Science, Department of Embryology, Baltimore, MD 21218, USA.
*Author for correspondence ([email protected])
Received 6 November 2014; Accepted 23 January 2015
which is evident from various Notch-related congenital diseases
and tumors in human patients (Koch and Radtke, 2010; Penton
et al., 2012) and in mice lacking Notch receptors or ligands in
various tissues (Conlon et al., 1995; Demehri et al., 2008; Gale
et al., 2004; Hamada et al., 1999).
Several methods have been developed and used to report Notch
activity, each with specific strengths and weaknesses [reviewed by
Ilagan et al. (2011)]. Taking advantage of the fact that the intracellular
domain of Notch (NICD) requires ligand-induced proteolysis to be
released from the cell membrane, we have previously described the
creation and analysis of a Notch1 activity-trap mouse line, Notch
intramembrane proteolysis-Cre or N1IP::Cre (Vooijs et al., 2007)
(Fig. 1A,E). In this knock-in line, the coding sequence for phage
recombinase Cre, which was followed by six myc tags (6MT) and
preceded by a nuclear localization signal (NLS), was targeted into one
Notch1 allele within exon 28, encoding the transmembrane domain of
Notch1 (Fig. 1E). This targeting strategy resulted in the expression of
a Notch1-NLSCre6MT fusion protein from this locus, in which the
intracellular domain of Notch1 is completely replaced by NLSCre6MT.
The binding of the ligand to cells expressing Notch1 and Notch1NLSCre6MT fusion protein triggers the release of NICD and the
NLSCre6MT protein, respectively. Whereas the NICD permits normal
development, NLSCre6MT, once inside the nucleus, could catalyze the
removal of any floxed allele in the genome. If a reporter is present, Cre
activity will ‘trap’ the cell and permanently mark its descendent lineage
independent of any further Notch1 activation (Fig. 1A). We have used
this line to show that this activity-trap strategy could identify lineages
experiencing Notch1 activation (Vooijs et al., 2007).
Despite our success, this NIP-Cre reporter line is relatively
insensitive and seemed to predominantly trap cells in which high or
repeated activation cycles are known to occur (e.g. endothelium),
while missing cells in many tissues known to rely on Notch1
activity [e.g. the hematopoietic system, (Vooijs et al., 2007)]. We
speculated that this could be caused by less-efficient transcription
from the manipulated allele and reduced activity by the insertion of
the 6MT (Vooijs et al., 2007). It was also hypothesized that, in some
tissues, sequences required for proper trafficking of Notch to the
membrane were lost when NICD was replaced with Cre.
Here, we report that removing the 6MT and adding an additional
polyadenylation signal significantly improved sensitivity, allowing
us to trap cells experiencing lower Notch activation thresholds.
When compared with the original N1IP-Cre reporter (now
designated as N1IP::CreLowActivity or N1IP::Cre LO), this new line
(N1IP::CreHighActivity or N1IP::Cre HI allele) traps Notch1 activation
in most tissues known to rely on Notch1, and identifies novel cells
utilizing Notch1 during their development and/or maintenance,
some of which will be reported elsewhere. Importantly, biochemical
analysis indicates that this new allele is cleaved as efficiently as
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DEVELOPMENT
Zhenyi Liu1,2, Eric Brunskill3, Scott Boyle2, Shuang Chen2,3, Mustafa Turkoz2,3, Yuxuan Guo2,4, Rachel Grant2
and Raphael Kopan2,3,*
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Development (2015) 142, 1193-1202 doi:10.1242/dev.119529
Notch1, supporting our previous observation that Notch trafficking
relies on signals coded in the extracellular domain (Liu et al., 2013).
This trap line, N1IP::CreLO and the related sibling N1IP::CreERT2
(Liu et al., 2009; Pellegrinet et al., 2011) line, are powerful new
tools for mapping alteration in Notch1 activation in vivo as a
consequence of genetic or pharmaceutical intervention.
RESULTS AND DISCUSSION
Biochemical analyses of N1IP::Cre constructs demonstrate
that a 6MT C-terminal fusion reduces Cre activity five- to
tenfold
N1IP::Cre mice contain a 6MT fused to the C-terminus of the Cre
recombinase protein (Fig. 1A,E). Similar Cre fusions described
elsewhere include CreErt2, in which a tamoxifen-responsive
domain controls nuclear entry of Cre (Feil et al., 1996; Zhang
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et al., 1996). Cre activity is reliant on Tyr 324, located 20 amino
acids from the C-terminus (Guo et al., 1997). Given the low labeling
index of N1IP::Cre in some tissues (Vooijs et al., 2007), we tested
whether this fusion might reflect a negative interference with
Cre-recombinase enzymatic activity. To test this hypothesis, we
developed an in vitro system to quantify Cre activity in primary
cells. First, we prepared reporters for Cre activity by isolating mouse
embryonic fibroblasts (MEFs) from embryos heterozygous for the
RosaR26R allele (Soriano, 1999). In these cells, β-galactosidase
(lacZ) will be expressed upon Cre-mediated recombination of a
single locus (Fig. 1C). Second, we constructed plasmids encoding
Cre and Cre-6MT containing a nuclear localization sequence
(NLS), followed by an internal ribosomal entry site (IRES) and
firefly luciferase (Luc, Fig. 1B). In a given transfected cell
population, lacZ activity will be a measurable surrogate for
DEVELOPMENT
Fig. 1. Creation of N1IP::CreHI. (A) Principle of cleavage-dependent Notch reporter. The replacement of Notch intracellular domain with Cre recombinase leads
to the release of Cre, but not of NICD, from the cell membrane after ligand-induced S2 and S3 cleavage. Cre will activate reporters, such as Rosa lacZ or
Rosa EYFP, which will remain active in all descendants. (B-D) Comparison of the enzymatic activity of Cre and Cre6mt fusion proteins. (B) Structure of the
plasmids used in the analysis. (C) Schema of MEF preparation and testing. (D) Comparison of β-gal activity in R26R-MEF cells expressing similar levels of
luciferase (note that the data are plotted on a log scale). (E) Comparison of N1IP::CreLO and N1IP::CreHI targeted locus. ANK, ankyrin repeats; LNR, Lin-Notch
repeat; TM, transmembrane domain; PEST, proline/glutamic acid/serine/threonine-rich motif; 2pA and 3pA, 2× and 3× polyadenylation signal. (F) Comparison of
mRNA levels among Notch1, N1IP::CreLO and N1IP::CreHI in 9.5-day embryos. (G) Comparison of released Cre levels between N1IP::CreLO and N1IP::CreHI in
newborn kidneys by western blot using anti-V1744 antibody. (H) Quantification of released Cre relative to the released N1ICD in the same sample.
recombinase activity within the population, whereas luciferase
activity will report relative output from the Cre-expressing plasmids
(transfection efficiency), independent of their recombinase activity.
When MEFs were transfected with increasing DNA concentrations,
luciferase activity scaled linearly throughout the entire range (from 7.8
to 500 µg, supplementary material Fig. S1), demonstrating that Cre
and Cre-6MT were expressed at similar levels. However, β-Gal
activity in extracts prepared from transfected cells indicated that
NLSCre6MT recombinase activity was significantly diminished
relative to Cre at every concentration (supplementary material
Fig. S1). Notably, measurable β-Gal activity could be detected at
the lowest transfected Cre concentration and continued to increase in a
relatively linear fashion, whereas NLSCre6MT activity increased only
above 60 ng (supplementary material Fig. S1). To compare Cre
activity at similar protein concentrations, we transfected the MEFs
with plasmid concentrations yielding identical photon flux and
measured β-Gal activity (Fig. 1D). Even at this high plasmid
concentration a sevenfold difference was noted between Cre and
Cre6MT (Fig. 1D). These results indicate that our original strain,
N1IP::Cre mice (Vooijs et al., 2007) and, most likely, the related line
N1IP::Cre ERT2 (Pellegrinet et al., 2011), under-report Notch activity
in vivo. This conclusion applies to all CreErt2 lines driven by weak
promoters, and could explain the differences in labeling observed
when different Cre fusion proteins are expressed from the same locus
as in Grisanti et al. (2013).
The creation of N1IP::CreHI reporter mice
An unfused activity-trap reporter might be able to trap Notch1 activity
at lower thresholds missed with the Cre6MT protein. To generate
mice with improved Notch-trap ability, we modified our targeting
constructs to create a new line of knock-in reporter mouse, N1IP::creHI
(Fig. 1E; supplementary material Fig. S2). First, we removed the
6Myc tag from the targeting construct to restore the original
C-terminus. Second, we added an additional polyadenylation signal
(AAUAAA) to the SV40 early polyadenylation sequence following
Development (2015) 142, 1193-1202 doi:10.1242/dev.119529
the Cre ORF, thus matching the total number of polyadenylation
signals (three) found in the wild-type Notch1 transcript. Finally, we
substituted a cytidine for an adenine at position 5178 of exon 28,
corresponding to a silent mutation at the third position for L1726 in
Notch1 (Fig. 1E; supplementary material Fig. S2). This offers a
simple, fast and non-radioactive method to pre-screen embryonic stem
cells (ESCs) for homologous recombination with pyrosequencing
(Liu et al., 2009), and enabled us to compare the abundance of mRNA
produced by each allele (Liu et al., 2013, 2011). We expected these
modifications to increase the relative abundance of the N1IP::CreHI
mRNA and to fully restore Cre activity.
To directly compare the mRNA level of N1IP::CreHI and
N1IP::CreLO, we mated Notch1+/CreHI (N1IP::CreHI heterozygote)
to Notch1+/CreLO (N1IP::CreLO heterozygote) mice and collected
E9.5 Notch1CreHI/CreLO embryos. These animals have no functional
NICD and do not survive beyond E10.5. Notch1+/CreHI embryos
served as a control. From these, we purified total RNA for RT-PCR
and performed pyrosequencing to compute the ratio of adenine
(N1IP::CreHI) to cytidine (wild-type Notch1 in Notch1+/CreHI
embryos or N1IP::CreLO in Notch1CreHI/CreLO embryos) at
nucleotide 5178 in exon 28. The results show that the addition
of the extra polyadenylation signal increases the stability of
N1IP::CreHI mRNA ∼twofold, to a level comparable to that of
the endogenous Notch1 [Fig. 1F; (Liu et al., 2011)]. To compare the
amount of the Cre protein that is released from the cell membrane
after ligand binding from the N1IP::CreHI and N1IP::Cre LO
proteins, we performed western blot on wild type, N1IP::Cre HI
heterozygote and N1IP::Cre LO heterozygote newborn kidneys with
an antibody against cleaved Notch1 (V1744), thus normalizing the
level of Cre to the NICD produced from the cleaved endogenous
Notch1. Consistently, we found a twofold increase in the abundance
of cleaved Cre relative to Cre6MT (Fig. 1G,H).
Importantly, because N1IP alleles are cleaved as effectively as
the endogenous Notch1 allele in the kidney (Fig. 1G,H), they
establish unequivocally that sequences in the intracellular domain
Fig. 2. Comparison of the labeling
pattern between N1IP::CreHI and
N1IP::CreLO at different embryonic
stages. N1IP::CreLO; RosaR26R/R26R
(A-D) and N1IP::CreHI; RosaR26R/R26R
(E-M) embryos at different stages stained
with X-gal. White arrowheads, notochord;
yellow arrows, intersegmental arteries;
black arrowhead in F, ventral half of the
developing CNS. (I) Sagittal section of an
E15.5 N1IP::CreHI; RosaR26R/R26R embryo
stained with X-gal. J and K show coronal
sections of E9.5 and E10.5 embryos,
respectively. Black arrowheads, notochord.
(L) Enlarged view of an E10.5 embryo.
Black arrows, intersegmental arteries.
(M) Stained sagittal section of the head of a
newborn pup. Thyroid gland is labeled with
a white arrow.
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Development (2015) 142, 1193-1202 doi:10.1242/dev.119529
beyond the cleavage site do not play a significant role in regulating
the trafficking of Notch proteins to the proper apical membrane
domain. This extends the conclusion derived from kidney epithelia
(Liu et al., 2013) to the kidney endothelial cells, the main
contributor of Notch1 ICD to whole kidney extracts. Whether this is
the case in all metazoans or in all mammalian cell types remains to
be determined.
Comparing N1IP::CreHI and N1IP::CreLO in vivo
N1IP::CreHI; RosaR26R/R26R sires were mated with RosaR26R/R26R
dams (Soriano, 1999) to trap cells experiencing Notch1
activation. Consistent with the data showing increased Cre
protein level and activity, we found that, throughout early
embryonic development, N1IP::CreHI embryos were more
intensely labeled by whole-mount β-Gal staining than N1IP::
CreLO embryos (4°C overnight, Fig. 2). Consistent with what we
observed in vitro, N1IP::CreHI-trapped cells experienced Notch1
activity earlier than N1IP::CreLO at all embryonic stages (Fig. 2).
Under the same X-gal development conditions, N1IP::CreHI;
RosaR26R/R26R embryos contained many labeled cells in the heart
(Fig. 2E, asterisk) and the notochord (Fig. 2E, white arrowhead;
Fig. 2J, black arrowhead) by E9.5. By contrast, no signal was
detected in N1IP::CreLO; RosaR26R/R26R embryos before E10.5,
when the only labeled cells were found in the heart [Fig. 2A,B;
(Vooijs et al., 2007)]. At E10.5, more cells were labeled in
N1IP::CreHI hearts (Fig. 2F, asterisk) and most, if not all, intersegmental arteries (Fig. 2F, yellow arrows; Fig. 2L, black arrows).
This indicates that Notch1 is activated earlier than E10.5 in
arterial endothelial cells, consistent with genetic observations
and phenotypes (Gridley, 2010). By contrast, the inter-segmental
arteries were sparsely labeled in the N1IP::CreLO mice at E11.5
(Fig. 2C). Accordingly, N1IP:CreHI/flox trans-heterozygotes, in
which the activated N1IP::Cre excises the floxed Notch1 allele,
suffer embryonic lethality at E10.5 due to the developmental
failure of the vascular system in the yolk sac and the embryo (Liu
et al., 2011), similar to the result of early endothelial cell-specific
conditional knockout of Notch1 (Limbourg et al., 2005) with
either one of two available Tie2-Cre strains (Kisanuki et al., 2001;
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Koni et al., 2001). By contrast, all N1IP:CreLO/flox transheterozygotes survive without discernible phenotype to P50
(Liu et al., 2011). Together, these data suggest that N1IP::CreHI
reports the activation of Notch1 in the endothelium with high
fidelity, trapping nearly all cells, whereas N1IP::CreLO reported
activation only in cells experiencing substantially higher Notch
activation levels (Vooijs et al., 2007). Interestingly, removal of
ADAM10 with Tie2-Cre (Kisanuki et al., 2001) was not lethal
(Zhao et al., 2014), perhaps allowing sufficient activity to persist
through an early, crucial developmental window. The use of these
lines to elucidate the role of signal strength in endothelial and
hematopoietic lineage separation from hemogenic endothelium
will be described elsewhere.
At E15.5, staining of sagittal sections showed widespread
labeling in the neural tube, brain, retina, major blood vessels,
intestine and vertebrae (Fig. 2I), correlating well with the reported
role of Notch1 in these tissues. At P0, a majority of the brain is
labeled and nerve fibers in the head region can be clearly identified
with β-Gal staining (Fig. 2M). Recently, it has been suggested that
Notch signaling regulates thyrocyte differentiation (Carre et al.,
2011; Ferretti et al., 2008). Accordingly, we observed widespread
labeling in the thyroid gland at P0 (Fig. 2M, white arrow).
To assess the behavior of N1IP::CreHI with a different reporter
strain, we crossed N1IP::CreHI and N1IP::CreLO with RosaeYFP/eYFP
mice (Srinivas et al., 2001). We collected kidneys at post-natal day 28
( p28) and compared the labeling patterns in the kidney cortex.
The entire CD31+ vascular tree within glomeruli is labeled in
both N1IP::Cre lines [Fig. 3, arrows; see also (Liu et al., 2013)],
consistent with the robust activation of Notch1 during the
development and maintenance of endothelial cells (Gridley,
2010). Despite the activation of Notch1 in the proximal tubule
epithelium during kidney development and its known contribution
to the maturation of these cells (Surendran et al., 2010a,b), very little
labeling is detected within this compartment in N1IP::CreLO;
Rosa+/eYFP mice. By contrast, a majority of proximal tubule
epithelia are Notch1-lineage positive in N1IP::CreHI; Rosa+/eYFP
mice (Fig. 3, arrowheads), consistent with its role in the
development and in regulation of tubule diameter (Liu et al.,
DEVELOPMENT
Fig. 3. 6Myc-Tag compromises Notch1-Cre activity in vivo. Kidneys were isolated from P28 N1IP::Cre LO; Rosa+/eYFP (A-E) and N1IP::CreHI; Rosa+/eYFP
(F-J), and examined for lineage-positive cells in the kidney cortex. Both N1IP::CreLO and N1IP::CreHI label CD31+ endothelial cells within glomeruli
(white arrows) and in vessels (asterisks in A-C, not shown in F-H). In N1IP::Cre LO mice, the associated tubular epithelium (Aqp1 positive; D,E) surrounding
glomeruli infrequently contains labeled cells (white arrowheads). By contrast, a majority of tubular epithelial cells in N1IP::CreHI mice are labeled (I,J). Scale bars:
100 µm.
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Development (2015) 142, 1193-1202 doi:10.1242/dev.119529
Fig. 4. Notch1-Cre activity in the nephron of the adult kidney.
N1IP::CreHI activity in the distal nephron was detected by the
overlap of EYFP with Tamm–Horsfall staining (left panel, the thick
ascending limb of the loop of Henle); arrows indicate co-labeling of
EYFP and Tamm–Horsfall glycoprotein. Clcnkb staining (middle
panel) shows distal convoluted tubules (same pattern seen with
Cdh1+; CytoK8−). Only a few cells were positive for both Calbindin
and EYFP (distal tubule and connecting segment, right panel).
Scale bar: 50 µm. 20× magnification.
N1IP::CreHI labeling patterns in the reproductive organs
The Notch signaling pathway plays essential roles in mediating the
interactions between the stem cells and their niche in various organs,
including the reproductive organs in Drosophila (Kitadate and
Kobayashi, 2010; Song et al., 2007; Ward et al., 2006) and
C. elegans (Hubbard, 2007). Whereas in the Drosophila ovary the
Notch receptor is expressed in the niche, the receptor is required in
the germ line stem cells in C. elegans. In rodent ovaries, Notch2 is
expressed in granulosa cells and the Jag1 ligand is expressed in the
germ cells (Johnson et al., 2001; Vanorny et al., 2014; Xu and
Gridley, 2013). The ovary of sexually mature virgin N1IP::Cre HI;
Rosa+/R26R females showed ubiquitous labeling in granulosa cells
(Fig. 5F), suggesting that Notch1 acts with Notch2 in the ovary but
is not activated in the oocyte (Vanorny et al., 2014). Accordingly,
we did not observe germline inheritance of an active reporter in
offspring of N1IP::Cre HI; RosaR26R/R26R females.
The role of Notch in the rodent testis remains controversial. Some
reports suggest a possible role for Notch1 in the niche during
spermatogenesis (Dirami et al., 2001; Hayashi et al., 2001; Murta
et al., 2013), but functional studies could not demonstrate an
essential function for Notch1 (Batista et al., 2012; Hasegawa
et al., 2012). If N1IP::CreHI is activated in germ cells during
spermatogenesis, it will result in germline inheritance of an active
reporter, manifest in uniform lacZ expression (stained blue by
X-gal) in offspring of N1IP::Cre HI; RosaR26R/R26R fathers.
However, we only saw three blue embryos out of more than 100
embryos that we stained, suggesting that Notch1 activation in germ
cells during spermatogenesis is an infrequent event or too low to be
detected by this line. Examination of the adult testis revealed
extensive N1IP::Cre HI activation within Sertoli cells (as marked by
SOX9 and TUBB3; Fig. 5I), consistent with previous studies
showing transgenic Notch Reporter GFP (TNR-GFP) activation in
Sertoli cells during multiple developmental stages (Garcia et al.,
2013; Hasegawa et al., 2012). There was also N1IP::Cre HI
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2013; Surendran et al., 2010a). These data demonstrate that removal
of the 6MT and addition of a third polyA site result in higher
recombinase Cre activity in vivo, consistent with our in vitro
findings.
Although these patterns alleviate the concern that N1IP::
CreHI might over-report Notch1 activity, it is possible that a
reporter that recombines at lower Cre concentrations will
generate a false-positive signature of Notch1 activity. To
evaluate the potential for false positives, we examined the
labeling patterns in the most sensitive reporter line we have
available, Ai3 (Madisen et al., 2010). The Ai3 reporter
recombination and expression are highly efficient and served
to determine the false-positive labeling rates in the distal
tubules of the kidney, where Notch1 expression is too low to be
detected (Liu et al., 2013). In the Ai3 kidneys, a significant
number of distal tubule cells experienced N1 activation, but
very few connecting segment cells were labeled (Fig. 4). This
might indicate a physiological reality in which signal strength
was insufficient to divert some cells from assuming a distal fate,
or the result of background proteolytic release of Cre. The
decline in labeling frequency from the proximal tubules
(∼100%) to the thick ascending limb (62.29±5.2%) to the
connecting segment (11.18±1.8%) is more consistent with the
former possibility; the intense labeling of distal tubule is
consistent with the first.
These data demonstrate that the N1IP::CreLO allele only labels the
subset of cells that experience high and/or persistent Notch1 activation.
Removing the 6MT increased labeling efficiency in many tissues
where Notch1 has a known function. It is worth noting that, although
N1IP::CreHI exhibits vastly improved sensitivity over N1IP::CreLO,
not all known Notch-mediated decisions are captured. For instance, we
did not observe labeling in the somite at E9.5 and E10.5 (Fig. 2E,F,J,
K), where Notch1 plays an important role at very low activation levels
(Conlon et al., 1995; Huppert et al., 2005; Lewis, 2003).
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Development (2015) 142, 1193-1202 doi:10.1242/dev.119529
Fig. 5. Notch1 labeling pattern in the
reproductive system. (A-E) Whole-mount
staining of gonads from male (A-C) and female
(D-E) embryos of indicated age and genotype.
(F) X-gal staining of an ovary from P53 virgin
R26R, N1IP::Cre HI female. (G,H) X-galstained section through epididymis from
immature (G) and mature (H) R26R, N1IP::
CreHI male. (I) Immunofluorescence staining
of adult N1IP::Cre HI; Rosa+/eYFP testis shows
high levels of Cre activity in Sertoli cells
identified with the Sertoli-specific markers
Sox9 and TUBB3. Note the colocalization of
EYFP with Sox9 or TUBB3. Scale bar:
100 µm. (J) Co-staining of K14 and EYFP in
different segments of the epididymis from
N1IP::Cre HI; Rosa+/eYFP male identifies
labeled cells as principal cells. (K) GFP+
epididymosome particles in the lumen of
epididymis from N1IP::Cre HI; Rosa+/eYFP male
are negative for DAPI. Ep, epididymis; Gd,
Gartner’s duct; Kd, kidney; Ms, metanephros;
Ov, ovary; Te, testis; Wd, Wolffian duct.
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vesicles that are derived from principal cells of the epididymis (Saez
et al., 2003). This is consistent with the idea that Notch1 activation
might not occur during spermatogenesis, but instead in the
epididymis, which is essential for the maturation of the sperm.
However, assignment of functional significance will necessitate
further studies.
N1IP::CreHI labeling pattern in the neural tissue
Notch activity is dispensable for establishing the neuroepithelium
as all Notch pathway mutants complete this process (de la Pompa
et al., 1997; Oka et al., 1995). We noted faint labeling in the
ventral half of the developing CNS (black arrow, Fig. 2F). To
identify the labeled cells we sectioned E10.5 embryos that had
been stained for β-Gal activity, and used a stereoscope to
photograph the slices (Fig. 6A-H). Two discrete progenitor
domains adjacent to the floor plate were strongly labeled in the
diencephalon (Fig. 6A), midbrain (Fig. 6B), hindbrain (Fig. 6C,D)
and spinal cord (Fig. 6E-H). Labeling of subpopulations of early-
DEVELOPMENT
activation within the interstitial compartment of the testis, probably
within vascular endothelial cells or perivascular cells, both of which
express Notch receptors and activate TNR-GFP during adulthood
(DeFalco et al., 2013).
By contrast, widespread labeling was detected as early as at E12.5
in the mesonephric tubules, the precursors of the epididymis
(Fig. 5A), persisting to the maturing epididymis (Fig. 5B,C).
The Wolffian duct in female embryos was labeled before it
degenerates into Gartner’s duct (Fig. 5E). Vasculature at the gonadmesonephros junction also was labeled (Fig. 5E). Sections of
newborn and adult epididymis showed that epithelial cells around
the lumen of all major segments, including the initial segment,
caput, corpus and tails are labeled, albeit at different frequencies
(Fig. 5G,H). Anti-K14 and EYFP double-staining of sections from
N1IP::Cre HI; Rosa+/eYFP mice showed that all labeled cells are K14negative principal cells (Fig. 5J). Stained particles in the lumen of
epididymis (Fig. 5K) were negative for DAPI staining, suggesting
that they are not sperm but rather epididymosomes, membranous
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Development (2015) 142, 1193-1202 doi:10.1242/dev.119529
born neurons in the dorsal aspect of the midbrain and hindbrain
was also detectable (Fig. 6B-D). This stripe pattern had been
observed previously with Dll1 and Jagged1 expression in the
chick hindbrain and neural tube (Marklund et al., 2010; Myat
et al., 1996). To align Notch activity stripes with its ligand we
crossed Dll1 +/lacZ (Hrabe de Angelis et al., 1997) mice with
N1IP::Cre HI; RosaeYFP/eYFP mice to generate E10.5 embryos
heterozygous for Dll1, Notch1 and Rosa-eYFP. Staining for lacZ
(Dll1 expression, Fig. 6I), EYFP (Cre activity) and Jagged1
confirmed that the cells experiencing Notch activation overlapped
with the ligand Dll1 (Fig. 6I). To identify the cells that
experienced high levels of Notch1 activity we stained a section
of the neural tube from N1IP::Cre HI; Rosa eYFP/eYFP mice with
Olig2 and Nkx2.2, which confirmed that progenitors in the pV2
and pV3 domains were labeled much more intensely than those in
the adjacent pMN and pV1 domains (Fig. 6J). We and others have
shown previously that Notch1 signaling was necessary to specify
neuronal subtypes, in particular V2 neurons and motor neurons in
the spinal chord (Del Barrio et al., 2007; Misra et al., 2014; Yang
et al., 2006). It would be interesting to investigate whether V2
neuron specification requires higher Notch1 signal strength than
other neuronal cell types.
In the CNS, neural progenitors undergo multiple rounds of
asymmetric division. Notch signaling oscillates to keep progenitors
cycling between committed progenitor state (Hes target gene
expression detected, proneural genes repressed) and pre-neuron
[ proneural gene expression detected, Hes1 repressed; (Imayoshi
et al., 2013; Kobayashi and Kageyama, 2010; Shimojo et al., 2008,
2011)]. After each division, the neuron daughter cell is thought to
activate Notch in its sister to reinforce the progenitor fate. A perfect
activity trap would label progenitors after the first Notch-dependent
decision, trapping all of their subsequent descendants (Fig. 7A).
Analysis of cortical layers from N1IP::CreHI; Rosa+/Ai3 (Fig. 7B;
supplementary material Fig. S3) shows co-labeling of EYFP
(N1IP::CreHI activity) and Foxp1 (a neuronal marker for late-born
upper layer neurons: Foxp1 labels neurons in layers II-IVa) in E15.5
cortical neurons (Fig. 7B,B′; supplementary material Fig. S3).
EYFP labeling was also detected in FoxP1–, earlier born neurons
and in stem/progenitor cells in the ventricular and subventricular
zones (VZ/SVZ) (supplementary material Fig. S4; arrows). By
contrast, we detected no EYFP labeling in Foxp1+ cortical neurons
of N1IP::CreLO; Rosa+/Ai3 embryos (Fig. 7C; supplementary
material Fig. S3). In addition, striatal projection neurons were
labeled very efficiently in N1IP::CreHI; Rosa+/Ai3 but only
marginally in N1IP::CreLO; Rosa+/Ai3(Fig. 7; supplementary
material Fig. S4). These data underscore the differences in
activation threshold between Cre and Cre6MT (and, by inference,
CreERT2).
The images in supplementary material Fig. S3 suggest that EYFP
labeling is denser in upper layer neurons than in lower layer neurons
and in progenitors located in the neocortex VZ/SVZ. Because
Notch1 signaling is thought to be activated in every asymmetric
progenitor division (de la Pompa et al., 1997; Imayoshi et al., 2010),
two possible scenarios could fit this pattern. First, a common
progenitor pool generates both lower layer (early) and upper layer
(late) neurons, and late-stage progenitors receive stronger Notch1
signals. Alternatively, the sensitive trap allele is still not sensitive
enough, and significant labeling requires gradual accumulation of
Cre protein over time, and thus favors late-stage progenitors that
produce upper layer neurons. We cannot rule out a third possibility
that separate progenitor pools contribute to lower and upper layer
neurons, with stronger signaling through the Notch1 receptor in the
latter.
Summary
Here, we demonstrate that Cre activity is compromised by fusion of
additional amino acids to the C-terminus. This observation implies
that all Ert2 fusion proteins are less active than Cre even when equal
nuclear concentrations are achieved, as shown in Grisanti et al.
(2013). The comparison between the two N1IP::Cre lines reveals
that Notch1 proteolysis, and thus signal strength, vary with time
within the same tissue (e.g. early- versus late-born cortical neurons,
1199
DEVELOPMENT
Fig. 6. Notch1-Cre activity in the developing CNS and neural tube defines a subset of neurons. (A-H) Whole-mount stained E10.5 N1IP::Cre HI, Rosa +/R26R
male embryos were cut into slices showing the diencephalon (A), midbrain (B), hindbrain (C,D) and spinal cord (E-H), and were photographed on a stereoscope.
(I) History of Notch activity detected with EYFP, Dll1 distribution with lacZ and Jagged1 expression with antibody staining in N1IP::CreHI; Rosa +/eYFP; Dll +/lacZ.
(J) EYFP, Olig2 and Nkx2.2 staining identifies EYFP+ progenitors as V2 neurons.
RESEARCH ARTICLE
Development (2015) 142, 1193-1202 doi:10.1242/dev.119529
Fig. 7. Notch1-Cre activity in neuronal precursors. Coronal sections through the telencephalic region at E15.5 detected EYFP staining as an indication of
Notch1-Cre activity in Rosa+/Ai3 developing neurons. (A) Schematic illustrating the likelihood that a precursor will be labeled by N1IP::CreLO or N1IP::CreHI. The
probability is depicted as a gradient. G, glia; ND, not detected. (B,C) The neuronal marker FoxP1 labels upper cortical layers (boxed regions in B′,C′) and
striatal projection neurons (boxed regions in B″,C″) in the N1IP::CreHI (B) and N1IP::CreLO (C). See supplementary material Fig. S3 for higher magnification view
of the boxed regions.
or anterior versus posterior stem cells along the intestine). Whether
the increase in signal strength is a consequence of unrelated biology
or an important requirement for the Notch signaling pathway
remains to be determined.
MATERIALS AND METHODS
Preparation of MEF cells
MEF cells were prepared under sterile conditions using a modified protocol
based on Takahashi et al. (2007) and Xu (2005). Briefly, RosaR26R/R26R dams
were crossed to a CD1 sire and embryos (Rosa+/R26R) were collected at E13.5.
Head, visceral organs and the urogenital system were removed. The remaining
tissues were pooled, washed three times with PBS and minced into ∼2 mm
pieces, using a sterile razor blade in a 100 mm diameter dish containing 20 ml
of warm Trypsin-EDTA (∼25 cuts per embryo). The cell mixture was pipetted
repeatedly with a 25 ml pipette and incubated at 37°C for 15 min. After
incubation, 10 ml of fresh Trypsin-EDTA was added, the tissue was further
dispersed using a 10 ml pipette and placed at 37°C for an additional 15 min.
The final cell mixture was transferred to a 50 ml conical tube, 20 ml of MEF
medium (DMEM, 10% FBS, 2 mM glutamine, 1000 U/ml penicillin and
1000 µg/ml streptomycin) was added to total volume of 50 ml, and spun down
at 1500 g for 5 min. The supernatant was discarded and the cell pellet
resuspended in 1 ml of MEF medium/embryo equivalent. One milliliter of
cell supernatant was plated in T75 flasks with 9 ml of MEF medium and
incubated overnight at 37°C in 5% CO2. The next day, cells were washed once
with PBS to remove dead/non-adherent cells and fresh medium was added.
Cells were frozen at passage 1. All experiments were performed on cells
expanded to passage 3 (P3).
Generation of N1IP::CreHI knock-in mice
In brief, the early SV40 polyadenylation sequence with one extra
artificially introduced polyadenylation signal (AATAAA) was cloned in
front of the FRT-flanked-neomycin selective cassette. The silent mutation
in L1726 was introduced with a site-directed mutagenesis kit (Stratagene)
into the SKB-L genomic clone that encompasses mouse Notch1 exons
26-32 (Vooijs et al., 2007). The SgrAI/SalI-digested NLScre fragment
and the SalI/BglII-digested SV40 polyadenylation sequence-FRT-neo
cassette were then cloned into SKB-L through the SgrAI/BamHI site to
replace the last 105 bases of exon 28 and to delete exons 29 and 30
( please note that BamHI and BglII share a compatible cohesive end, and
after ligation the BamHI site is lost) (supplementary material Fig. S2).
This plasmid was then sequenced and linearized with EcoRI for ESC
electroporation.
ESC targeting and screening
We targeted the Notch1 locus using 129X1Sv/J-derived SCC10 ESCs (for
further information regarding this line see http://escore.im.wustl.edu/
SubMenu_celllines/wtEScell_linesSCC10.html) and selected for G418resistant ESC colonies. Cells were first pre-screened with pyroscreening,
utilizing the C5178A polymorphism (Liu et al., 2009), and those with a C/A
ratio between 40 and 60% at this position were further screened by both longrange PCR and Southern blot to confirm bona fide homologous recombinants
[HR, (Liu et al., 2009); supplementary material Fig. S2)]. Positive HRs were
karyotyped, and one line (ES190) was injected into C57B6 blastocysts. The
resulting chimera male mice were mated to wild-type C57B6 female mice. F1
males were mated to FRT deleter mice (Rodriguez et al., 2000) to remove the
FRT-flanked neomycin cassette.
Transfection of MEF cells
1200
Western blot
Western blot was performed as described in Liu et al. (2013). Rabbit
monoclonal anti-cleaved Notch1 (anti-V1744, Cell Signaling) antibodies
were used at 1:1000 dilution.
X-gal staining
X-gal staining was performed as described in Vooijs et al. (2007). Briefly,
embryos or freshly dissected tissues were fixed in 4% PFA in 1× PBS
supplemented with 2 mM MgCl2 on ice for 1-2 h and washed with 1× PBS
+2 mM MgCl2. For whole-mount staining, embryos were developed in Xgal staining solution at 4°C overnight; for frozen section staining, tissues
were immersed in 30% sucrose in 1× PBS+2 mM MgCl2 overnight and
DEVELOPMENT
For transfection, 3.75×104 cells/well P3 MEFs were plated in the
afternoon before transfection in 24-well tissue culture plates with 500 µl
of MEF medium (without penicillin/streptomycin). Transfection was
performed using the Lipofectamine LTX system (Invitrogen) according to
the manufacturer’s protocol. For each well, 0.5 µg total DNA (with the
empty pCS2 vector added to reach the final concentration), 3.25 μl of LTX
reagent and PLUS reagent at a 1:1 ratio with DNA were used. The
transfection mixture was incubated with the cells at 37°C for 18 h and
replaced with fresh MEF medium. In control experiments using GFP
plasmids, ∼70% transfection efficiency was observed. Firefly luciferase
and β-gal activity were measured 24 h later, as previously described
(Ilagan et al., 2011).
embedded in optimal cutting medium (OCT, Tissue-Tek) and stained in Xgal staining solution at 37°C overnight or until signal was detected.
Immunohistochemistry
Immunohistochemistry was performed as described in Liu et al. (2013).
Briefly, frozen sections were blocked with normal donkey serum and then
incubated with primary antibodies at 4°C overnight. Following three times
washing with 1× PBS, the sections were incubated with fluorescenceconjugated secondary antibodies, washed with 1× PBS and mounted for
imaging. The following primary antibodies were used: chicken anti-GFP
(1:500; Aves Lab, GFP-1020) or rabbit anti-GFP (1:1000; Invitrogen,
A11122), rabbit anti-Foxp1 (1:2000; Abcam, ab16645), mouse antiCalbindin (1:500; Sigma-Aldrich, C9848), rabbit anti-Tamm–Horsfall
protein (THP) (1:250; Biomedical Technologies, BT-590), rabbit antiClcnkb (1:200; GeneTex, GTX47709), rabbit anti-olig2 (1:500; Millipore,
AB9610), goat anti-Nkx2.2 (1:50; Santa Cruz Biotech, SC-15015), chicken
anti-K14 (1:200; a generous gift from Dr Julie Segre, National Human
Genome Research Institute, National Institutes of Health), goat anti-Jag1
(1:100; Santa Cruz Biotech, SC-6011), rabbit anti-β-galactosidase (1:5000;
Cappel Labs/MP Biomedicals, 08559761), rat anti-CD31 (1:100; BD
Bioscience, 550274), goat anti-aquaporin-2 (1:200; Santa Cruz Biotech,
SC-9882), rabbit anti-Sox9 (1:4000; Millipore, #AB5535) and mouse antiTUBB3 (TUJ1) (1:1000; Covance, #MMS-435P). Images were acquired
with either ApoTome2 (Zeiss) or a Nikon 90i upright wide-field
microscope. Images were processed using Nikon Elements software,
Photoshop or Canvas.
Acknowledgements
We thank members of the Kopan laboratory for many fruitful discussions and Ms
Mary Fulbright for expert technical assistance. Special thanks to Drs Tony De Falco
(reproductive system), Kenny Campbell and Masato Nakafuku (central nervous
system) for expert advice and for critical reading of the manuscript.
Competing interests
The authors declare no competing or financial interests.
Author contributions
Z.L. and R.K. conceived the project and wrote the manuscript; Z.L., E.B., S.B., S.C.
designed and performed experiments; M.T., Y.G. and R.G. performed experiments.
Funding
Y.G. was supported fully, and R.K., Z.L., S.C. and M.T. were supported in part by the
National Institutes of Health (NIH) [R01GM55479 awarded to R.K.]. S.B. and R.G.
were supported by the NIH [R01DK066408 awarded to R.K.]. R.K. and E.B. were
supported in part by the William K. Schubert Chair for Pediatric Research. Deposited
in PMC for release after 12 months.
Supplementary material
Supplementary material available online at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.119529/-/DC1
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