The contribution of Notch1 to nephron segmentation in the developing kidney
is revealed in a sensitized Notch2 background and can be augmented by reducing
Kameswaran Surendrana, Scott Boylea, Hila Baraka, Mijin Kimb, Colin Stomberskia,
Brent McCrightc, Raphael Kopana,⁎
aDepartment of Developmental Biology, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, MO 63110, USA
bDepartment of Organismal Biology and Anatomy, University of Chicago 1027 E 57th Street, Chicago, IL 60615, USA
cDivision of Cellular and Gene Therapies, Center for Biologics Evaluation and Research Food and Drug Administration, NIH Building 29B, Room 2NN13, 20 Lincoln Dr., Bethesda,
MD 20892, USA
a b s t r a c ta r t i c l e i n f o
Received for publication 2 September 2009
Revised 22 October 2009
Accepted 9 November 2009
Available online 13 November 2009
We previously determined that Notch2, and not Notch1, was required for forming proximal nephron
segments. The dominance of Notch2 may be conserved in humans, since Notch2 mutations occur in Alagille
syndrome (ALGS) 2 patients, which includes renal complications. To test whether mutations in Notch1 could
increase the severity of renal complications in ALGS, we inactivated conditional Notch1 and Notch2 alleles in
mice using a Six2-GFP::Cre. This BAC transgene is expressed mosaically in renal epithelial progenitors but
uniformly in cells exiting the progenitor pool to undergo mesenchymal-to-epithelial transition. Although
delaying Notch2 inactivation had a marginal effect on nephron numbers, it created a sensitized background
in which the inactivation of Notch1 severely compromised nephron formation, function, and survival. These
and additional observations indicate that Notch1 in concert with Notch2 contributes to the morphogenesis of
renal vesicles into S-shaped bodies in a RBP-J-dependent manner. A significant implication is that elevating
Notch1 activity could improve renal functions in ALGS2 patients. As proof of principle, we determined that
conditional inactivation of Mint, an inhibitor of Notch-RBP-J interaction, resulted in a moderate rescue of
Notch2 null kidneys, implying that temporal blockage of Notch signaling inhibitors downstream of receptor
activation may have therapeutic benefits for ALGS patients.
© 2009 Elsevier Inc. All rights reserved.
The mammalian kidney forms as a result of reciprocal inductive
interactions between the metanephric mesenchyme (MM) and the
ureteric bud. MM cells undergo mesenchymal-to-epithelial transi-
tion (MET) and through a series of stereotypical morphogenetic
events form nephrons composed of distinct tubular segments along
a proximal–distal axis. The cellular processes that convert a spherical
aggregate of mesenchymal cells into a tubular structure composed of
distinct simple epithelial cell types and the molecules that orches-
trate these processes are only beginning to be identified (Dressler,
2006). In mice, Notch2, a type-I transmembrane protein, plays an
essential role early in nephron segmentation to ensure the emer-
gence of proximal cell fates (Cheng et al., 2007; Kopan et al., 2007).
In humans, reduced Notch activity results in Alagille syndrome,
observed in patients with one mutant allele of the Notch ligand
Jagged1 (ALGS1, Piccoli and Spinner, 2001) or the receptor Notch2
(ALGS2 (McDaniell et al., 2006). The four mammalian Notch genes
code for receptors that mediate short-range communication via a
conserved mechanism. Notch1, Notch2, and genes coding for their
ligands Dll1 and Jag1 are all expressed in the renal vesicle (RV), the
first structure to display epithelial characteristics during MET (Chen
and Al-Awqati, 2005; Cheng et al., 2007; Piscione et al., 2004).
Following ligand binding, a juxtamembrane domain unfolds, permit-
ting metalloproteases to cleave and remove the Notch extracellular
domain. Ectodomain shedding is followed by intramembrane
cleavage mediated by γ-secretase; this step releases the Notch
intracellular domain (N-ICD) to translocate into the nucleus where it
associates with the transcription factor RBP-J and recruits the
transcription activation machinery (Kopan and Ilagan, 2009). In
the nucleus, N-ICD may need to compete for binding to RBP-J with
ubiquitous co-repressor proteins including Mint (Oswald et al.,
2002; Tsuji et al., 2007; Yabe et al., 2007). Although Notch1 and 2
and the ligands Jag1 and Dll1 could act redundantly in kidney
development, inactivation of Notch2, γ-secretase, or RBP-J in the
MM prior to RV formation results in complete absence of glomeruli
and proximal tubules (PT), with only distal tubules forming; these
structures are formed, however, in the absence of Notch1 (Cheng
Developmental Biology 337 (2010) 386–395
⁎ Corresponding author. Fax: +1 314 747 5503.
E-mail address: firstname.lastname@example.org (R. Kopan).
0012-1606/$ – see front matter © 2009 Elsevier Inc. All rights reserved.
Contents lists available at ScienceDirect
journal homepage: www.elsevier.com/developmentalbiology
et al., 2003; Cheng et al., 2007; Wang et al., 2003). This is despite the
fact that Notch1 is activated in the RV and N1-ICD remains easily
detectable in the presumptive precursors of PT and podocytes within
the S-shaped bodies.
Since both N1-ICD and N2-ICD have the same affinity for RBP-J
(Del Bianco et al., 2008; Friedmann et al., 2008; Lubman et al., 2007)
and overexpression of N1-ICD can drive renal epithelial progenitors
to the PT cell fate (Cheng et al., 2007), it remained possible that
Notch1 made a contribution to the overall level of canonical Notch
signaling within the RV and S-shaped bodies, one that was too small
to support nephron segmentation in the absence of Notch2.
Alternatively, endogenous Notch1 is activated but incapable of
activating the same target genes that Notch2 activates during
nephrogenesis at physiologic levels. Finally, Notch1 activation may
lag behind Notch2, missing the developmental window (Chen and
Al-Awqati, 2005). To test which of these alternatives explain our
results and to delay Notch2 inactivation until after onset of kidney
development, we removed conditional Notch2 alleles using the
Six2-GFP::Cre transgene. This allowed nephrons to form with limiting
amounts of Notch2 and provided a sensitized, functional background
on which we could assess the contribution of Notch1 to kidney
development. We determined that Notch1 contributed to nephro-
genesis in vivo, albeit to a lesser degree than Notch2. Since Notch1
was capable of activating the same target genes as Notch2, we asked
if, in principle, methods aimed at augmenting Notch1 signaling could
help ALGS patients. We inactivated Mint, an inhibitor of N-ICD-
mediated transactivation of Notch target genes (Oswald et al., 2002;
Tsuji et al., 2007; Yabe et al., 2007) and asked if PT or glomeruli
reemerged in the complete absence of Notch2. Modest improvement
was indeed seen in Notch2 null kidneys, suggesting that efforts at
elevating Notch1 activity in ALGS2 patients with one functional
Notch2 allele, or in ALGS1 patients with global reduction in Notch
signaling, may have therapeutic benefits.
Materials and methods
All experiments involving mice were approved by the Washing-
ton University IACUC. The Six2-GFP::Cretgtransgenic mice (Park et
al., 2007) were bred with mice with floxed alleles of RBP-J (RBP-Jf/f)
(Tanigaki et al., 2002), Notch1 (N1f/f) (Yang et al., 2004), Notch2
(N2f/f), or both N1f/f;N2f/f. Pax3-Cretg; N2+/f(Cheng et al., 2007)
were bred with mice with floxed alleles of Mint (Mintf/f)(Yabe et al.,
2007). Compound heterozygotes were bred with mice homozygous
for the floxed alleles of the desired genes. All mice used in this study
were maintained on mixed backgrounds. Mice and embryos were
et al., 2003), primer sequences are available upon request, and their
age was estimated based on the assumption that noon of the day on
which a vaginal plug was observed was embryonic day 0.5 (E0.5).
Analysis of serum blood urea nitrogen levels was done at the O'Brein
Center renal chemistry core and presented as milligram per deciliter.
Histology and immunohistochemistry
For analysis of GFP expression pattern, the kidneys were
dissected and frozen in OCT embedding media on dry ice. Frozen
blocks were sectioned at 6- to 12-μm thickness and air-dried. The
sections were then rinsed in PBS and fixed for 5 min in 4% PFA
before visualization of GFP or were stained for Pax2 (1:200,
Covance) followed by Cy3-conjugated secondary antibody and then
counterstained with DAPI before visualization of fluorescent signals.
For visualization of Six2 and GFP, kidneys were fixed in 4% PFA for
30 min and then rinsed in 30% sucrose overnight before embedding
in OCT. For all immunohistochemistry, kidneys were fixed
overnight in Bouin's fixative at 4 °C, except 4% paraformaldehyde
was used for detection of RBP-J, washed, and stored in 70% ethanol
prior to embedding in paraffin and sectioning at 5- to 7-μm
Prior to immunostaining, the sections were de-paraffinized, boiled
for 20 min in Trilogy (Cell Marque) or Antigen Unmasking Solution
(Vector Labs H-3300 for detecting RBP-J), and cooled at RT for 20 min.
Detection of RBP-J and Notch2 required quenching of endogenous
hydrogen peroxidase activity with 3% H2O2treatment for 10 min at
RT. Sections were then blocked in PBS containing 1% bovine serum
albumin (BSA), 0.2% powdered skim milk, and 0.3% Triton X-100 for
30 min at RT prior to incubation primary antibody overnight in a
humidified chamber at 4 °C. For anti-RBP-J staining (Cosmo Bio Co.,
SIM-2ZRBP2, 1:100), the sections were additionally blocked with
avidin and then biotin (Vector Labs SP-2001) prior to incubation with
primary antibody. Tissue sections were then incubated with secon-
dary antibody conjugated with biotin (1:500), then developed using
ABC Vectastain kit (Vector Labs, PK-6100) followed by tyramide
amplification (PerkinElmer, NEL744001KT).
For detecting Notch2 tissue expression pattern, we used an anti-
body raised against the 258 carboxy-terminal amino acids of Notch2
(Varadkar et al., 2008) (1:100), tissue sections were permeabilized
with 1% Triton X-100 in PBS for 15 min at RT prior to blocking with
50 mM Tris pH8.0, 150 mM NaCl containing 5% donkey serum, and
0.05% Tween 20. After overnight incubation with primary antibody
overnight, the sections were developed by incubating with HRP-
conjugated secondary antibody and then by tyramide amplification.
Additional primary antibodies and lectins utilized in the study
include: Six2 (1:500; HM6123 was a gift from Andrew McMahon
(Kobayashi et al., 2008)), GFP (1:500, AVES Laboratory Inc.), anti-
beta-gal (1:5000; Cappel), p21 (1:200; Santa Cruz Sc-6246), NCAM
(1:200; Sigma), Jagged-1 (1:100; Santa Cruz Sc-6011), WT-1 (1:100;
Santa Cruz Sc-192), and FITC-Conjugated Lotus Lectin (1:200; Vector
Laboratories). Secondary antibodies conjugated with Cy3 (Jackson
ImmunoResearch) or Alexa488 (Molecular Probes) were utilized to
visualize the binding pattern of primary antibodies.
Estimation of glomerular number and density
The optical fractionator probe of the Stereo Investigator system
(MicroBrightField, Inc.) was utilized to estimate glomerular number
and kidney volume. Every 20th kidney section was analyzed, and
counting was performed under 10× magnification of hematoxylin-
and eosin-stained sections. Glomerular density was calculated by
dividing the estimated number of total glomeruli by the estimated
total volume for each kidney.
Verification of Notch2 floxed allele deletion
E14.5 Six2-GFP::Cretg; N2f/fkidneys were digested by incubating
with 2.25% pancreatin and 0.7% trypsin in Ca-Mg free Tyrode's
solution (pH 7.4) for 1 h on ice, while periodically disrupting the
kidneys by repeated pipetting. DMEM with 10% fetal bovine serum
was added, and samples were then filtered through a 36-μm nylon
mesh (Small Parts; #CMN-0035D) and were centrifuged at 800 rpm
for 3 min. The pellet was resuspended with FACS sorting buffer
(containing 3% FCS in PBS with 0.01% azide). GFP+cells were
sorted using a MoFlo high-speed flow cytometer (Dako Cytomation,
Fort Collins, CO). Genomic DNA was extracted from GFP+cells
using the QIAamp DNA Micro kit (Qiagen). Thirty nanograms of
genomic DNA was used with the following primers to amplify and
detect any undeleted Notch2 floxed alleles: ttcaaccccagataggaag-
cagctcagctcacag and gtgcactggagttgggggacccataacttcg. Serial dilution
of N2+/fgenomic DNA with wild-type DNA was used to verify that
we are able to detect as little as one intact Notch2 floxed allele per
K. Surendran et al. / Developmental Biology 337 (2010) 386–395
All results are presented as mean±SD. In the graphs, the height
of the bar represents the mean and the error bars represent
standard deviation. Two-tailed unpaired t-tests were performed to
compare mice with Notch-deficient kidneys to their wild-type
littermates. The resulting p values are mentioned in the text and
The Six2-GFP::Cre transgene is expressed mosaically in the renal
epithelial progenitor population, with uniform high level of expression
in the induced mesenchyme and pretubular aggregates prior to
We have previously observed that canonical Notch2 signaling
was required for proximal nephron development during a narrow
developmental window at or before the conversion of the renal
vesicle (RV) to an S-shaped body (SB), and that Notch1 was dis-
pensable (Cheng et al., 2007). We hypothesized that if we could
inactivate Notch2 just prior to RV formation, we would create a
Notch2-deficient environment in which to test if Notch1 contributed
to nephron development. The Six2-GFP::Cre BAC transgene (Cheng
et al., 2007; Humphreys et al., 2008) had unique properties making
it ideal for this experiment. This transgene was designed for
expression in the nephron progenitor population, within the
metanephric mesenchyme (MM) (Kobayashi et al., 2008). The self-
renewing progenitors for all renal epithelial lineages, except for the
collecting ducts, reside in the mesenchymal cap condensate (MCC)
surrounding each ureteric bud tip and can be recognized by the
expression of Six2, Cited1, and Pax2 (Boyle et al., 2008; Kobayashi
et al., 2008; Mugford et al., 2009; Self et al., 2006). These cells reside
in two broad sub-domains: cortical to the branching ureteric bud the
‘capping metanephric mesenchyme’ (CMM; Six2+, Cited1+, Wnt4−)
contains a pure stem population, and lateral to it the ‘induced
metanephric mesenchyme, (IMM; Six2low, Cited1−, Wnt4+) is a
transient nephron precursor population beginning to respond to
Fig. 1. The Six2-GFP::Cre transgene is expressed mosaically in renal epithelial progenitor population, with uniform high level of expression in the induced mesenchyme and
pretubular aggregates prior to RV formation. (A–A″) Six2-GFP::Cre transgene (green) is expressed at variable levels within renal epithelial progenitors. Not all Six2+cells (red)
express GFP::Cre at E13.5 (arrowheads). GFP::Cre is expressed at higher levels in cells exiting orno longer in the definitive renal epithelial progenitor pool(arrows) and inpretubular
aggregates (⁎) and continues to be expressed in RV (⁎⁎). A′ is a higher magnification of the boxed area in panel A. (B and B′) Co-staining for Pax2 (red) and the Six2-GFP::Cre
transgene (green) revealsthat not allmesenchymal capcondensatesthat are Pax2+express Crerecombinase(blue arrowheads) at P1. The white arrowheads are placed as positional
references and point at cells expressing both Pax2 and GFP. (C) Importantly, due to strong expression of Cre in pretubular aggregates, all cells achieved recombination of floxed
alleles by the S-shaped stage as demonstrated by β-galactosidase expression in every S-shaped body cell of E13.5 Six2-GFP::Cretg;Rosa26R kidneys. All cells of the S-shaped bodies
(arrowheads) have experienced cre-mediated recombination and have turned on β-galactosidase expression at E13.5. All scale bars are 100 μm.
K. Surendran et al. / Developmental Biology 337 (2010) 386–395
Wnt9b signals (Mugford et al., 2009). Staining for GFP::Cre and for
endogenous Six2 protein revealed that in the CMM, many Six2-
expressing cells did not express GFP::Cre at E13.5 (arrowheads in
Figs. 1A and A′). A higher fraction of IMM cells, lateral to the
ureteric bud, stained for both Six2 and GFP (Figs. 1A, A′, and A″).
Importantly, cells exiting the progenitor pool during MET, which
down-regulate Six2 expression (Mugford et al., 2009), retained
high levels of GFP::Cre expression (arrows in Figs. 1A and A″).
Furthermore, GFP remained detectable in pretubular aggregates
ventral (medullary) to the branching ureteric bud (asterisks in Figs.
1A and A″). Fate mapping of the Wnt4+population, which includes
the IMM and pretubular aggregates, revealed that these cells
quickly convert to nephron fates and are not the self-renewing
nephron progenitors (Kobayashi et al., 2008). Expression of the
Six2-GFP::Cre transgene faded in the RV (asterisks in Fig. 1A′). As
late as postnatal day 1 (P1), MCC contained Pax2+, GFP−
progenitors (Figs. 1B and B′), suggesting that this Cre line would
allow for the retention of intact Notch2 floxed alleles (N2f/f) within
the renal epithelial progenitor population of Six2-GFP::Cretg;N2f/f
mice. Thus, although some progenitor cells become depleted of
floxed alleles within the MCC early on and would be expected to
generate Notch2-depleted descendents throughout nephrogenesis,
this mosaically expressed Six2-GFP::Cre transgene allows many
MCC to retain floxed alleles until the end of nephrogenesis.
Importantly, due to strong expression of Cre in pretubular
aggregates, all cells achieved recombination of floxed alleles by
the SB stage as demonstrated by β-galactosidase expression in
every SB cell of E13.5 Six2-GFP::Cretg;Rosa26R kidneys (Fig. 1C,
Kobayashi et al., 2008).
Delayed inactivation of Notch2 in renal epithelial progenitors results
in sufficient nephron segmentation and proximal tubule formation
Based on this characterization of the Six2-GFP::Cre BAC
transgene, we hypothesized that using this line, we could attain
genetic inactivation of Notch2 as late as in the pretubular
aggregates of each round of nephrogenesis, a stage in which all
cells are GFP::Cre-positive, while retaining sufficient numbers of
stem cells with an intact Notch2 locus for subsequent rounds of
nephrogenesis. Six2-GFP::Cretg;N2+/fmale mice were mated with
N2f/ffemales to generate Six2-GFP::Cretg;N2f/fmice. In contrast to
Pax3-Cretg;N2f/fanimals that die at birth due to absence of PT and
glomeruli (Cheng et al., 2007), Six2-GFP::Cretg;N2f/fmice had a
normal lifespan and were fertile. Histological examination on P1
confirmed that all newborn Six2-GFP::Cretg;N2f/fkidneys had many
mature nephrons containing glomeruli with Wilms' tumor 1
(WT1)-positive podocytes connected to lotus tetragonolobus lectin
(LTL)-positive PT (Fig. 2D). In addition, the presence of numerous
SB in the cortex at P1 suggested that many Notch2-containing stem
cells survived to birth (Figs. 2E and F). Nonetheless, examination of
several P1 Six2-GFP::Cretg;N2f/fkidneys revealed many mice (∼30%
of n=23) that had visibly smaller kidneys when compared with
control littermates (n=13; Figs. 2A and B). Smaller kidneys had on
average 62% fewer glomeruli than the wild-type littermate kidneys
(Fig. 2C; 4950±488 in N2f/f, n=3 versus 1902±1038 in Six2-
GFP::Cretg; N2f/f, n=3, p=0.001), and glomerular density was
reduced by 49% (2.4±0.2×10−6glomeruli/μm3in N2f/f, n=3
versus 1.2±0.8×10−6glomeruli/μm3in Six2-GFP::Cretg;N2f/f, n=3,
p=0.002). Reduced glomerular number and density would be
expected if some stem cells lost Notch2 protein, generating
Notch2-deficient RV that failed to form nephrons containing
proximal segments; variability in nephron numbers would be
expected if the number of stem cells expressing the transgene was
variable in this outbred cohort. Dependence of kidney phenotypes
on genetic background has been previously reported (McCright
et al., 2002). Alternatively, since inherent fluctuations in biochemi-
cal reactions will increase the probability of failure when involving
low copy number molecules (Levine et al., 2007), limiting amounts
of Notch2 could fail to support proximal development in a sto-
Staining for Notch2 protein in N2f/flittermate kidneys detected
protein in the pretubular aggregates, the RV and the SB (Fig. 2G).
In contrast, staining of Six2-GFP::Cretg;N2f/fP1 kidneys could not
detect Notch2 protein in any nephron precursors (Fig. 2F). To
confirm the timing of Notch2 inactivation, we isolated GFP-
positive cells (enriched for pretubular aggregates; Fig. 1) by FAC
sorting from E14.5 Six2-GFP::Cretg; N2f/fMCC (Fig. 2H). Whereas
we could easily detect an intact, floxed Notch2 allele when
present at 1 copy per 100 cells (Fig. 2I, lane 4), we could not
detect any intact Notch2 alleles in the GFP-positive population
(Fig. 2I, lanes 10 to 12). This indicates that by MET, we achieved
complete inactivation of Notch2.
In conclusion, this particular Six2-GFP::Cre transgene allowed
many progenitors to postpone Notch2 inactivation until MET. Notch2
mRNA/protein synthesized prior to gene inactivation in the Six2-
GFP::Cretg;N2f/fline may allow sufficient—though undetectable—
Notch2 to act alone (or in conjunction with other Notch receptors)
within the epithelial lineages to support normal segmentation in
most of the developing nephrons.
Notch1 is required in Six2-GFP::Cretg;N2f/fmice for nephrogenesis
The survival of Six2-GFP::Cretg;N2f/fmice allowed us to ask if
Notch1 contributed to PT and podocyte determination in this
reduced Notch2 signaling background. If Notch1 failed to compen-
sate for loss of Notch2 because of temporal differences in expression
(Chen and Al-Awqati, 2005) or because of qualitative differences
between Notch1 and Notch2, then Notch1 would not be expected to
activate required Notch2 transcriptional targets, and inactivation of
Notch1 in Six2-GFP::Cretg;N2f/fkidneys should not compromise the
kidney any further. Conversely, the dominant role of Notch2 could
be due to a quantitative difference between the two receptors.
Attaining the level of Notch activity in the RV necessary for nephron
segmentation in this sensitized background could require a contri-
bution from Notch1 (Fig. 5B). In this scenario, combined inactivation
of Notch1 and Notch2 should further compromise nephrogenesis.
Whereas Six2-GFP::Cretg;N1f/fmice were all born with wild-type-like
kidneys with multiple PT and glomeruli (Figs. 3A and B), removal of
one Notch1 allele in a Notch2-deficient background severely compro-
mised nephrogenesis (Fig. 3C). At P1, 67% of Six2-GFP::Cretg;N1+/f;
N2f/fmice have visibly smaller kidneys than wild type, twice the
frequency of what we observed in Six2-GFP::Cretg;N2f/fmice (31%,
Fig. 5A). Only one-third of the Six2-GFP::Cretg;N1+/f;N2f/fmice sur-
vived to weaning, and most of these mice died by 6 months of age
(Fig. 3D). Survivors displayed a variable reduction in kidney size, the
longest surviving mice containing at least one near-normal kidney
(Fig. 3E). In extreme cases, only a few glomeruli and PT formed,
some of which were dilated (Fig. 3C). Combined complete
inactivation of Notch1 and 2 (Six2-GFP::Cretg; N1f/f; N2f/f) resulted
in pups that died at P1 and all kidneys had few PT with little (or no)
evident glomeruli (Fig. 5A, Supplementary Fig. S1). Compromised
nephron segmentation was evident by the presence of abnormal
NCAM+, Pax2+ structures in the nephrogenic zone of Six2-GFP::
Cretg;N1+/f;N2f/fkidneys (Fig. 3H) where comma and S-shaped
structures are found in wild-type kidneys (Fig. 3G). We analyzed
blood urea nitrogen (BUN) levels in surviving Six2-GFP::Cretg;N1+/f;
N2f/f, Six2-GFP::Cretg; N2f/f, and Six2-GFP::Cretg;N1f/fand compared it
to wild-type littermates within the first week after birth. Only Six2-
GFP::Cretg;N1+/f;N2f/fmice had compromised renal function at this
age (49.8±35.2 mg/dl, n=14, compared with 25±3.1 mg/dl in
controls, n=19, p=0.0045; Fig. 3F).
K. Surendran et al. / Developmental Biology 337 (2010) 386–395
The data presented above suggest a quantitative difference
exists between Notch1 and Notch2 activity during the early stages
of nephrogenesis but demonstrate that both provide the same
function. Further support to this hypothesis comes from compar-
ing wild-type, Pax3-Cretg;N2+/f(data not shown), or Six2-GFP::
Cretg;N1f/fmice (all wild type in appearance) with Six2-GFP::Cretg;
N1f/f;N2+/fmice. Forty percent of Six2-GFP::Cretg;N1f/f;N2+/fmice
have visibly smaller kidneys at P1 (n=10) when compared with
Fig. 2. Delayed inactivation of Notch2 in the renal epithelial progenitors results in sufficient nephron segmentation and proximal tubule formation for survival. Compared with N2f/f
littermate kidneys (A), approximately 30% of postnatal day 1 Six2-GFP::Cretg; N2f/fkidneys (B) are visibly smaller. (C) Graph illustrating the average (height of the bar) number of
glomeruli in N2f/f(n=3) is 4950±488 versus 1902±1038 in the visibly smaller Six2-GFP::Cretg; N2f/f(n=3, p=0.001) P1 kidneys. (D) All the kidneys of newborn Six2-GFP::Cretg;
N2f/fmice have many mature nephrons containing podocytes within glomeruli (red) and LTL+proximal tubules (green). (E) P1 Six2-GFP::Cretg; N2f/fkidneys contain NCAM+
(green) and Pax2+(red) S-shaped bodies confirming that nephron segmentation occurred. (F) The kidneys of newborn Six2-GFP::Cretg; N2f/fmice have no detectable Notch2 protein
in the nascent renal epithelial structures, whereas in (G) N2f/fkidneys, Notch2 protein (red) co-localizes with NCAM (green) in the cell membrane of a subset of cap condensate cells
and all nascent renal epithelial structures. (H) By FACS we isolated the GFP+ population (boxed by dashed line) from E14.5 Six2-GFP:: Cretg; N2f/fkidneys. (I) PCR analysis of genomic
DNA extracted from GFP+FACS sorted cells reveals that the Notch2 floxed alleles are inactivated in the GFP+cells. We determined the lower limit of detection of the Notch2 floxed
allele to be one copy per 100 cells. Lanes 1 and 13 contain 100 bp DNA ladder (Invitrogen), lanes 2 to 12 contain PCR product, in which the starting template for lanes 2 to 6 contain
10-fold serial dilutions of 30 ng of genomic DNA extracted from tail of N2+/fmouse diluted in wild-type genomic DNA. The starting template for lane 7 is wild-type genomic DNA; for
lane 8, is water; for lane 9, is 30 ng of genomic N2f/fDNA; and for lanes 10, 11, and 12, is 30 ng of genomic DNA extracted from GFP+kidney cells of three different Six2-GFP:: Cretg;
N2f/fmice. The arrow at the left hand side of gel points at the expected size of the Notch2 floxed allele. We are able to detect a strong PCR product even when only one Notch2 floxed
allele ispresent per 100 genomes (lane 4) but not inthe GFP+cells (lanes 10, 11, and12). Scale bars in panels Aand B are 1 mm, scale bar in panel Dis 100 μm, andscale bars in panels
E, F, and G are 20 μm. UB: ureteric bud, S: S-shaped body, V: blood vessel.
K. Surendran et al. / Developmental Biology 337 (2010) 386–395
wild-type mice (Fig. 5A and Supplementary Fig. S1). This result
also demonstrates that like in humans, a single Notch2 allele is
not sufficient on its own to provide all RV with the required
activity. The dominant function for Notch2 in nephrogenesis is
also reflected in the observation that Six2-GFP::Cretg;N1f/f;N2+/f
mice have a milder reduction in nephrogenesis when compared
with Six2-GFP::Cretg;N1+/f;N2f/fmice (Fig. 5A and Supplementary
Inactivation of RBP-J resembles the combined inactivation of Notch1
and Notch2 with a severe reduction in S-shaped body formation and
development of proximal tubules and glomeruli
The experiments described above established that Notch1 and
Notch2 contribute to nephrogenesis but did not resolve why
Notch1 plays a minor role relative to Notch2. One possibility is that
although both receptors contribute to nephron segmentation, they
Fig. 3. Inactivation of Notch1 in the Notch2-deficient kidneys reveals that Notch1 is required for nephrogenesis, normal renal function, and survival. WT1 and LTL staining of
postnatal day 1 (A) wild-type (N1+/f; N2+/f), (B) Six2-GFP::Cretg;N1f/f, and (C) Six2-GFP::Cretg;N1+/f;N2f/fkidneys reveals reduced number of proximal tubules with the inactivation
of one Notch1 allele in the Notch2-deficient background. (D) The graph depicts the percentage of mice of a genotype surviving as they age up to 6 months. Whereas the Six2-GFP::
Cretg; N2f/fmice (n=10) have a normal lifespan (red dashes), the Six2-GFP::Cretg;N1+/f; N2f/fmice (n=16) have a variably reduced lifespan (blue line) and Six2-GFP::Cretg;N1f/f;
N2f/fmice (n=4) die soon after birth (green dashes). (E) Bisected kidneys from one Six2-GFP::Cretg;N1+/f; N2f/fmouse that died at 6 months revealed one hydronephrotic
obstructed kidney (top panel) and a second hypoplastic kidney (bottom panel). (F) The blood urea nitrogen (BUN) levels are increased in Six2-Cretg; N1+/f;N2f/fmice within the first
week of birth when compared with wild-type littermates (49.8±35.2 mg/dl, n=14, compared with 25±3.1 mg/dl in controls, n=19, ⁎p=0.0045). The average BUN levels are
represented by the height of each bar and one standard deviation by the error bars. (G) In postnatal day 1 kidneys of N2f/fmice NCAM and Pax2 staining revealed comma and S-
shaped structures in the nephrogenic zone. (H) Abnormal nephron segmentation was evident by the presence of abnormal NCAM+, Pax2+ structures (arrowhead) in the
nephrogenic zone of Six2-GFP::Cretg;N1+/f;N2f/fkidneys. All scale bars are 100 μm.
K. Surendran et al. / Developmental Biology 337 (2010) 386–395
do so in different ways. For instance, only Notch2 functions via
RBP-J while Notch1 may function in a RBP-J-independent manner.
Alternatively, there may be unique inhibitors of N1-ICD that
dampen the ability of N1-ICD to associate with RBP-J. To ask if
canonical or non-canonical signals were involved, we inactivated
RBP-J, the common mediator of the canonical Notch signaling
pathway, using the Six2-GFP::Cretgtransgene. If only Notch2 signals
via RBP-J, Six2-GFP::Cretg;RBP-Jf/fmice would be expected to produce
a similar phenotype to Six2-GFP::Cretg;N2f/fmice. If, however,
Notch1 made its contribution to nephron segmentation via RBP-J,
then Six2-GFP::Cretg;RBP-Jf/fmice should have very few nephrons
and would be comparable to the combined inactivation of Notch1
All Six2-GFP:; Cretg;RBP-Jf/fmice die within 2 days of birth with
abnormally small kidneys and compromised vasculature (Fig. 4A
versus D). A few nephrons containing glomeruli and PT did form
(Figs. 4G and H). These few nephrons were capable of filtration and
connected to the collecting duct system as the bladders contained a
clear filtrate (arrow in Fig. 4D); however, filtration was insufficient,
resulting in death. RBP-J is normally expressed in the nucleus of all
cells in the nephrogenic zone (Fig. 4B) and the cytoplasm of mature
PT at P1 (Fig. 4C). Except for a few cells (arrowhead in Fig. 4E),
most MCC cells Six2-GFP::Cretg;RBP-Jf/fkidneys were deficient for
RBP-J (Fig. 4B versus E) at P1, and the few PT that formed did not
express RBP-J (Fig. 4F). The presence of a few RBP-J+MCC is likely
due to the mosaic expression of Six2-GFP::Cretgwithin the MCC. The
cortexes of P1 Six2-GFP::Cretg;RBP-Jf/fkidneys were devoid of SB
and mature nephrons, abundant in wild-type littermates (arrows in
Fig. 4B). Furthermore, expression of Jag1, a marker for the
presumptive PT precursors, was absent from E17.5 Six2-GFP::Cretg;
RBP-Jf/fkidneys except for the spotted expression in RV-like
structures (Supplementary Fig. S2). The similarity between Six2-
Fig. 4. Inactivation of RBP-J resembles the combined inactivation of Notch1 and Notch2 with a severe reduction in S-shaped body formation and development of proximal tubules and
glomeruli. Compared with (A) wild-type, RBP-Jf/fmice (D), the Six2-GFP:: Cretg; RBP-Jf/fmice die by postnatal day 2 and although their kidneys are small, managed to produce a clear
filtrate present in the bladder (black arrow). (B) In RBP-Jf/fmice, the RBP-J protein (red) is present in the nucleus of all kidney cells at P1 including S-shaped bodies (arrows), which
results in (C) mature LTL+ proximal tubules containing RBP-J. (E) In Six2-GFP:: Cretg; RBP-Jf/fP1 kidneys, only a few mesenchymal cap condensate cells still retain RBP-J expression
(white arrowhead) and (F) the absence of RBP-J protein in the proximal tubules. (G and H) The Six2-GFP:: Cretg; RBP-Jf/fkidneys develop very few glomeruli (yellow arrowhead) and
proximal tubules (blue arrowheads or green signal). Scale bars are 100 μm.
K. Surendran et al. / Developmental Biology 337 (2010) 386–395
GFP::Cretg;RBP-Jf/f(Fig. 4) and Six2-GFP::Cretg;N1f/f;N2f/fkidneys
(Supplementary Fig. S1) indicates that both Notch1 and Notch2
signal via RBP-J during nephrogenesis. In aggregate, Notch1 and
Notch2 activate the canonical Notch signaling pathway to con-
tribute to PT and glomeruli formation, with partial redundancy and
paralog dominance: Notch2 plays a dominant role but Notch1 pro-
vides activity via RBP-J, which becomes essential when Notch2
amounts are limiting (Figs. 5A and B).
Fig. 5. The specification of proximal nephron cell fates is acutely sensitive to the dosage of canonical Notch1 and Notch2 signaling activity, which allows the removal of repressors of
RBP-J-dependent transcription to partially rescue nephrogenesis. (A) The frequency of animals with normal kidney size, y-axis, inversely correlates with the increasing loss of
canonical Notch signaling activity, x-axis. The height of each blue bar represents the frequency of animals of a particular genotype having normal kidney size at P1. The number of
mice analyzed per genotype at P1 is indicated next to the genotype. All floxed alleles (f) presented in the graph were inactivated using Six2-GFP:: Cretg. Wt=wild-type littermates.
(B) A model of the individual contributions of Notch1 and Notch2 to nephron segmentation based on current and previous observations. Notch2 signaling (orange bar) is critical for
reaching the threshold of Notch signaling activity (dashed line) required for sufficient PT and podocyte (POD) development to ensure survival. Inactivation of Notch2 in the Six2-
GFP::Cretg; N2f/fmice creates a sensitized Notch signaling background in which a further reduction in Notch signaling by inactivating Notch1 to create Six2-GFP::Cretg;N1+/f; N2f/f
mice reveals that both Notch1 and Notch2 are required for PT and POD, in the absence of which only distal tubule (DT) form. (C, D) Progressive loss of Mint on the Pax3-Cretg; N2f/f
background results in the reemergence of proximal cell fates. (C) Kidneys from Pax3-Cretg; N2f/f, Pax3-Cretg; Mint+/f; N2f/f, and Pax3-Cretg;Mintf/f; N2f/fwere scored on a rank-order
scale (see Figure S3) for degree of rescue based on the presence of LTL+PT structures. Decreasing doses of Mint consistently corresponded to a greater degree of PT reemergence.
⁎p=0.001. ⁎⁎p=0.0008. (D) Representative (average rank-score) images of sagittal, center cut P0 kidney sections from each of the above genotypes stained withWT1(red; MM and
podocytes) and LTL (green; PT). In some cases, we observed WT1+ and LTL+ cells polarized in the same structure in Pax3-Cretg; Mintf/f; N2f/fmice (inset), which we have never
observed in Pax3-Cretg; N2f/fanimals.
K. Surendran et al. / Developmental Biology 337 (2010) 386–395
Removal of Mint, a repressor of RBP-J-dependent transcription, mildly
rescues PT formation in Pax3-Cretg;N2f/fkidneys
Since Notch1 is activated at high levels during nephron segmen-
tation relative to other areas in the developing embryo, we wished to
examine if, in principle, augmenting Notch1 activity could benefit
humans with one functional allele of Notch2 or Jag1. Nuclear N-ICD/
RBP-J complexes can be inhibited by several endogenous repressor
proteins, including SMRT, CIR, KyoT2, and Mint/SHARP/Spen (Kopan
and Ilagan, 2009). Of these, Mint is present in the kidney (Newberry
et al., 1999), it antagonizes canonical Notch activity, and its removal
was shown to elevate Notch activity in vivo (Kuroda et al., 2003;
TanigakiandHonjo, 2007; Tsuji et al., 2007; Yabe et al.,2007). To ask if
reducing inhibitor activity might improve N1-ICD activity, we
inactivated Mint in Pax3-Cretg;N2f/fmice. Because Pax3-Cretg;N2f/f
mice have no remaining nephrons, and we have shown that Notch1
does contribute to the formation of normal nephrons, any enhance-
ment of N1-ICD activity achieved by removing Mint in these mice
should allow at least a few glomeruli and PT to form. Furthermore, PT
should be readily detectable given that this background completely
lacks them. On the other hand, if N1-ICD is functioning at full capacity
during nephron segmentation and the limiting factor is the amount of
N1-ICD, we would expect no rescue of PT formation in Pax3-Cretg;N2f/f
mice. Consistent with the hypothesis that Notch1 activity can be
augmented by removal of inhibitors, deletion of conditional Mint
alleles on the Notch2 null background (Pax3-Cretg;Mintf/f;N2f/fmice)
resulted in a reemergence of some nephrons containing LTL-positive
PT structures and WT1-positive cells resembling glomerular podocyte
(Fig. 5C and D). This “rescue” was consistent, as improvement over
Pax3-Cretg;N2f/fmice was observed in 12 of 13 double knockout
animals, but variable since not all animalsdisplayed similar number of
nephrons. Even the best rescue was not sufficient for a functional
kidney, resulting in perinatal death of all Pax3-Cre;Mintf/fN2f/fmice.
Interestingly, even inactivation of one copy of Mint resulted in the
emergence of some PT in Notch2 mutant mice (Pax3-Cretg;Mint+/f;
N2f/f) suggesting a dose response. In order to quantify the degree of PT
formation (as marked by LTL+ structures) in Pax3-Cretg;N2f/fmice
with progressive loss of Mint, we devised a rank-order score, with no
PT (Pax3-Cretg;N2f/f) defined as 1, and the greatest degree of rescue
observed defined as 5 (Supplementary Fig. S3). Four observers
blinded to genotype information scored Pax3-Cretg;N2f/fkidneys con-
taining 2, 1, or 0 copies of Mint. The scores for each genotype were
averaged and the t-test analysis established if the scores were
statistically significant. This analysis confirmed a dose-dependent
increase in LTL+PT as more Mint alleles were inactivated in the
absence of Notch2 (Fig 5C). Pax3-Cretg;N2f/f(average rank score
1.125) improved with one Mint allele removed (Pax3-Cretg;Mint+/f;
N2f/f, average rank score 2.0) and more so with two alleles inactivated
(Pax3-Cretg;Mintf/f;N2f/f, average rank score 3.6). This proof of
principle experiment demonstrates that N1-ICD activity can be
increased to further its contribution to nephrogenesis and implies
that the functional activity of Notch1 can be enhanced with reagents
that free RBP-J of transcriptional repressors.
Paralog dominance: Notch1 and Notch2 contribute jointly but unequally
to nephron formation
We established that the BAC Six2-GFP::Cretgis not expressed in all
renal epithelial progenitors; it is expressed at higher levels in induced
metanephric mesenchyme and in renal progenitors exiting the stem
cell pool, a period during which it completely inactivates floxed
alleles. We used this unique tool to generate kidneys with reduced
Notch2 signaling by delayed inactivation of Notch2. Although Six2-
GFP::Cretg;N2f/fcells completely lost immunoreactivity to Notch2, the
exact timing of Notch2 protein depletion within this population is
impossible to determine and is likely variable. This resulted in some
nephron loss and in visibly smaller kidneys in 30% of newborn mice
without decreasing viability. We present evidence that in this com-
promised background, Notch1 activity was necessary to maintain
proper nephron segmentation and subsequent maturation. Inactiva-
tion of one Notch1 allele in Six2-GFP::Cretg;N1+/f; N2f/fkidneys
lowered Notch activity to or below this threshold, significantly
reducing the number of nephrons and thus compromising viability
in 60% of newborns. Inactivation of Notch1 and Notch2 resembled loss
of RBP-J in Six2-GFP::Cretg; RBP-Jf/fmice and resulted in a fully
penetrant, lethal small kidney phenotype. Our analysis of an allelic
series progressively reducing canonical Notch signaling capacity
during nephrogenesis leads us to propose a model in which each
nephron segments properly if overall Notch activity crosses a
threshold level in the RV. The Notch2 receptor is dominant (its loss
in the MM prior to the onset of kidney development results in
complete loss of nephrons), but Notch1 contributes to nephrogenesis
(its contribution becomes apparent when Notch2 levels are limiting).
In mice, Notch2 heterozygotes are close to the threshold, as evident
from the effects of reducing Jag1 (McCright et al., 2002) or Notch1
(this study) in a Notch2 heterozygous background. In humans, where
haploinsufficiency of Notch2 or Jagged1 has pathological conse-
quences, the activity levels presumably fall below this threshold
(McDaniell et al., 2006) even with a full complement of Notch1. Given
that each RV gives rise to exactly one nephron, the number of
nephrons that form thus equals the sum of RVs in which overall Notch
signals reached this threshold and accurately reflects the dose of
remaining wild-type Notch1 and Notch2 alleles. Why Notch2 is
dominant to Notch1 in this process and the molecular basis of this
threshold are under investigation.
The phenomenon of Notch paralog dominance occurs in humans,
where Notch1 is unable to compensate for mutations in Notch2 that
result in juvenile cystic kidney disease, one complication of Alagille
syndrome (Lu et al., 2003; McDaniell et al., 2006). In addition to the
kidney, Alagille syndrome impacts the heart, liver, and craniofacial
bone development, all sites where Notch1 and Notch2 overlap but
where Notch2 may be the dominant paralog (Geisler et al., 2008).
Inversely, loss of Notch1 (Rangarajan et al., 2001), but not Notch2 or
Notch3, compromises the skin barrier, resulting in sensitivity to
carcinogens; Notch1 activity seems equal to the combined output of
Notch2 and Notch3 (Demehri et al., 2009). In addition, Notch1 is the
dominant paralog in arterial endothelial cells (Krebs et al., 2000;
Vooijs et al., 2007), the aortic valve (Garg et al., 2005), oligoden-
drocyte (Givogri et al., 2002), osteoclast progenitors (Bai et al.,
2007), and T-cells, where Notch2 can only drive development if Dll4
is overexpressed (Besseyrias et al., 2007).
Elevating Notch transcriptional activity during nephrogenesis in ALGS
patients may ameliorate kidney disease
Even in the absence of any obvious developmental malformations,
one functional Notch2 allele in Six2-GFP::Cretg;N1f/f; N2+/fmice is not
sufficient to support a normal level of nephrogenesis as normal
nephrogenesis required a contribution from Notch1. In humans, the
relative contribution of Notch1 must be less than in mice given that
mutations in one Notch2 allele compromise kidney development.
Alternatively, the proteins produced by mutant Notch2 alleles in
ALGS2 patients function as dominant-negative receptors. We deter-
mined that Notch1 contributes to normal renal development in mice
and may do so in humans where only Notch2 and Jagged1 mutations
have been associated with defects in renal development. Notch1
mutations could act as modifiers of ALGS, increasing the severity of
Activation of a single Notch molecule generates a unit of signal in
the form of a released intracellular domain; a phenotypic threshold is
K. Surendran et al. / Developmental Biology 337 (2010) 386–395
reached when enough units reach the nucleus to replace the appro- Download full-text
priate and sufficient numbers of RBP-J containing transcriptional
repressor complexes with transactivating complexes. The factors that
could contribute (alone or in combination) to differences between
Notch1 and Notch2, and therefore to relative signal ‘strength’, are
under investigation in our laboratory. Although developing therapies
aimed at elevating Notch1 activity in ALGS is a rationale goal that
requires deeper mechanistic understanding to limit untoward effects,
a proof of principal experiment was attempted to demonstrate
feasibility of this approach. Towards this end, we report that gene-
tically ablating Mint, a known Notch inhibitor (Kuroda et al., 2003;
Tanigaki and Honjo, 2007; Tsuji et al., 2007; Yabe et al., 2007),
resulted in regaining some nephrons on a Notch2 null background.
This result indicates that generation of N1-ICD is not the only factor
limiting the contribution of Notch1 to nephrogenesis, and efforts
aimed at elevating Notch1 activity transiently downstream of
receptor activation (via agonists or inhibition of antagonist) in
ALGS2 patients with one functional Notch2 allele, or in ALGS1 patients
with global reduction in Notch signaling, may have therapeutic
benefits. The next task will be to demonstrate the benefit and safety of
such strategies in animal models of ALGS (McCright et al., 2002).
We thank the histology core of the Department of Developmental
Biology for preparation of tissue sections. We thank the Mouse Genetics
Core and the Digestive Diseases Research Core Center (supported by NIH
P30DK052574) for the generation of genetically altered mice. We thank
Mary Blandford and Tao Shen for assistance with animal husbandry and
mouse genotyping. We thank Dr. Tasuku Honjo for the Mint and RBP-J
William Eades and Jacqueline Hughes in the Siteman Cancer Center High
Speed Sorter Core Facility for performing cell-sorting segments of our
experiments. The Siteman Cancer Center is supported in part by NCI
Cancer Center Support Grant P30 CA91842. This work was supported by
National Institutes of Diabetes and Digestive and Kidney Disease
(DK066408) and the O'brien Center Grant (5P30DK07933). S.B. was
supported by institutional training grant (5T32DK007126).
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