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First published online 17 January 2007
doi: 10.1242/dev.02773
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1 Department of Molecular Biology and Pharmacology and Department of Medicine at
Washington University School of Medicine, 660 South Euclid Avenue, Campus Box
8103, St Louis, MO 63110, USA.
2 Department of Molecular and Cellular Biology, Harvard University, 16 Divinity
Avenue, Cambridge, MA 02138, USA.
3 Institute for Molecular Biology OE5250, Medizinische Hochschule Hannover,
Carl-Neuberg-Str. 1 D-30625 Hannover, Germany.
* Author for correspondence (e-mail: kopan{at}wustl.edu)
Accepted 5 December 2006
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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-secretase-independent form of
Notch1 intracellular domain drives the specification of proximal fates where
all endogenous, ligand-dependent Notch signaling is blocked by a
-secretase inhibitor. These results establish distinct (non-redundant),
instructive roles for Notch receptors in nephron segmentation.
Key words: Notch, Rbp-J, Wnt4, Proximal tubule, Podocytes, Nephron segmentation, Mouse
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Nephrons form from a simple epithelial precursor, the renal vesicle (RV),
itself a product of a Wnt-induced mesenchymal-to-epithelial transition in the
outer cortex (Carroll et al.,
2005
). The RV begins a series of molecular changes reflected by a
stereotyped set of morphological and molecular changes. Morphologically, the
RV transitions through a comma-shaped, then an S-shaped body stage before
fusing with the adjacent epithelium of the ureteric bud (UB)-derived
collecting duct system to establish a continuous tubular network. Asymmetric
expression of Brn-1 (Pou3f3 - Mouse Genome Informatics)
(Nakai et al., 2003),
E-cadherin (cadherin-1 - Mouse Genome Informatics) and cadherin 6
(Cho et al., 1998) provide some
of the first evidence for polarization of the proximodistal axis (the future
glomerulo-collecting duct axis), but it is not until the S-shaped body stage
that the future proximodistal axis is readily distinguishable. At this time,
Pax2 is highly expressed within the distal portion of the S-shaped body,
including the region that fuses to the UB. Podocyte precursors, which
differentiate into glomerular podocytes, reside in the proximal limb of the
S-shaped body (the visceral epithelial cells) and express high levels of
Wilms' tumor-1 (Wt1) (Kreidberg et al.,
1993). Adjacent to the podocyte in the cleft of the proximal limb,
the vascular endothelial network of the future glomerular filtration apparatus
starts to assemble. Although fate-mapping studies have not addressed the
contribution of distinct regions within the S-shaped body to the future
nephron, a cadherin-6-positive domain that lies between the presumptive
podocytes and the Pax2(+) distal tubule progenitors is likely to give rise, at
least in part, to the proximal convoluted tubule, a Lotus
tetragonolobus lectin (LTL) (Laitinen
et al., 1987)-binding epithelium in the mature nephron first
visible at embryonic day (E) 14.5-15.5 in the mouse. The process of nephron
formation continues at the periphery of the mouse kidney up to postnatal day
7; newborn kidneys thus contain nephrons at all stages of development.
Previous work examining the expression of Notch pathway components
(Chen and Al-Awqati, 2005;
Leimeister et al., 2003;
Piscione et al., 2004) and
modulating Notch signaling (Cheng et al.,
2003; Wang et al.,
2003) supported the argument for a Notch pathway activity in
mammalian nephrogenesis. Notch genes encode single-transmembrane receptors
that mediate short-range communication between cells. Receptor binding to
ligand expressed on adjacent cells triggers the shedding of its extracellular
domain and the subsequent cleavage of the transmembrane domain by the enzyme
-secretase (for a review, see Mumm
and Kopan, 2000). On -secretase-mediated proteolysis, the
Notch intracellular domain (N1-ICD or N2-ICD) is released and translocates to
the nucleus, where it associates with a DNA-bound REL-like protein (Cbf1/Rbp-J
in vertebrates; Rbpsuh - Mouse Genome Informatics) and promotes transcription
of its targets (Fryer et al.,
2004; Lubman et al.,
2004). In mammals, four Notch homologs
(Notch1-4) and at least five ligands [jagged 1 (Jag1), Jag2,
delta-like 1 (Dll1), Dll3 and Dll4] mediate these signaling events.
Notch1, Notch2, Dll1 and Jag1 mRNA are detected in the RV
and its derivative; the expression domain of Notch1 partially
overlaps with Notch2 in the S-shaped body
(Chen and Al-Awqati, 2005).
Notch2 and Jag1 are also expressed in the collecting duct. Humans
haploinsufficient for jagged 1 (Li et al.,
1997) are prone to Alagille syndrome, one symptom of which can
result in the development of renal abnormalities
(McCright, 2003;
Piccoli and Spinner, 2001),
whereas abnormal glomerulogenesis is observed when Notch2 activity is reduced
(McCright et al., 2001).
Notch3 expression has been reported in the distal portion of the
S-shaped body (Piscione et al.,
2004); however, a lacZ knockin into the Notch3 locus
indicates that only the glomerulus and blood vessels may express Notch3
(H.-T.C. and R.K., unpublished).
To date, no specific study has addressed the regional-specific action of
the Notch pathway in nephron patterning, although the general, organ-wide
inhibition of -secretase activity suggested that Notch activity is
likely to be important, as podocytes and proximal tubules are lost when
-secretase activity is abolished
(Cheng et al., 2003;
Wang et al., 2003). However,
the observed phenotypes cannot be unequivocally attributed to loss of Notch
signaling due to the existence of multiple other substrates of
-secretase.
Here we have addressed the specific function of Notch1 and Notch2 by tissue-specific modulation of their activity. These studies reveal distinct roles for Notch1 and Notch2 in nephron development. Notch2 activity is essential for patterning of the proximal regions of the nephron. However, although Notch1 is normally activated, and when overactivated is capable of proximalizing the nephron, it is not sufficient for the development of proximal cell fates. Thus, local activation of Notch2 during renal tubule morphogenesis is a central determinant of segmented pattern in the mammalian nephron.
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MATERIALS AND METHODS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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) to N2f/f females to obtain
Pax3-cretg/+; N2f/f embryos or pups. We crossed
Pax2-cretg/+; N1
1/+
(heterozygote for a null allele) (Conlon et
al., 1995) males to N1f/f females to produce
Pax2-cretg/+;
N1f/1 embryos.
Pax2-cretg/+;Rbp-Jf/+
(Ohyama and Groves, 2004;
Tanigaki et al., 2002) males
were crossed with Rbp-Jf/f females to produce
Pax2-cretg/+; Rbp-Jf/f embryos. Using the same
scheme, we also generated Pax2-cretg/+; N2f/f
pups, which had the same phenotype as Pax3-cretg/+;
N2f/f pups. In some cases, we analyzed
Pax2-cretg/+;
N1f/1;
Dll-1lacZ embryos (Hrabe
de Angelis et al., 1997).
Six2-GFP::Cre males were crossed to RosaNotch
(Notch1 intracellular domain) (Murtaugh et
al., 2003) females to obtain RosaNotch/+;
Six2-GFP::Cretg/+ embryos. All mice used in this study were
maintained on mixed backgrounds. Embryos were genotyped by standard PCR
protocol. Noon of the day on which a vaginal plug was scored was designated as
E0.5. The day when pups were born was designated as their first postnatal day
(P1).
Generation of Notch1-/- 
wild-type chimeric embryos
The procedure is described in detail in Hadland et al.
(Hadland et al., 2004).
Briefly, compound heterozygotes for the Rosa26 locus and N11
were crossed, blastocysts removed and cultured. LacZ-expressing control and
Notch1-deficient embryonic stem (ES) cells were derived from Rosa26/+ and
N1/1;
Rosa26/+ embryos, respectively. ES cells with normal karyotype were then
injected into E3.5 wild-type CD1 blastocysts, which were then transferred into
the uterus of pseudopregnant CD1 females. Whole-mount ß-galactosidase
(ß-gal) staining (described below) was performed on the kidneys harvested
from embryos at E16.5, before paraffin-embedded sectioning.
Metanephric organ culture
Mouse metanephric organ culture was performed as described by Rogers et al.
(Rogers et al., 1991).
Briefly, kidneys were removed from E12.5 mouse embryos and cultured on
transwell filters (Falcon, pore size 1 µm) at an air-fluid interface in a
serum-free medium consisting of equal volumes of Dulbecco's modified Eagle
medium and Ham's F12 medium containing 25 mmol/l HEPES, sodium bicarbonate
(1.1 mg/ml), 10 nmol/l Na2SeO3.5H2O,
10-11 M prostaglandin E1, and iron-saturated transferrin (5
µg/ml). Medium was refreshed every day and the metanephroi were cultured
for as long as 6 days.
Histology and immunohistochemistry
The kidneys or the cultured explants were fixed in Bouin's fixative or in
4% paraformaldehyde (PFA) [for LTL and 5-Bromo-2'-deoxyuridine (BrdU)
analysis], embedded in paraffin and sectioned at 5 µm. The sections were
then stained with Hematoxylin and Eosin (H&E) for histological analysis.
For immunohistochemistry, the sections were boiled in Trilogy (Cell Marque)
for antigen retrieval. The antibodies and the lectins were diluted as follows:
rabbit anti-mouse cadherin 6 (1:300; kindly provided by Dr Dressler)
(Cho et al., 1998), Ck8
(TROMA1; 1:10; Developmental Studies Hybridoma Bank), E-cadherin (1:1000;
Transduction Labs), Jag1 (1:200; Santa Cruz), N-Cam (1:300, Sigma), Pax2
(1:200; Covance), Wt1 (1:100, Santa Cruz), synaptopodin (1:40, gift from Dr P.
Mundel), laminin a1 and fluorescein isothiocyanate-conjugated LTL (1:100;
Vector Labs). Hoechst (0.5 µg/ml, Sigma) was used for nuclear staining.
Fluorescein- and Cy3-conjugated anti-IgG corresponding to the species of the
primary antibodies was used to visualize the antigen. For cadherin 6,
E-cadherin and Jag1, we used horseradish peroxidase (HRP)-conjugated IgG
followed by tyramide-conjugated FITC or Cy3 for better results. For Notch1
detection, metanephroi were fixed in Bouin's fixative after 6 days in organ
culture, dehydrated and embedded in paraffin wax. Sections (7 µm) were
boiled in Trilogy for antigen retrieval, pre-blocked with PBS supplemented
with 1% BSA, 0.2% skimmed milk, and 0.3% Triton X-100. A rabbit primary Notch
1 antibody (1:200, abcam ab27526) was used, followed by a biotinylated
anti-rabbit IgG (1:3000). The Vectastain ABC kit (Vector Laboratory, Inc) and
tyramide-conjugated Cy3 (TSA-Plus Cyanine 3, PerkinElmer, 1:100) were used to
detect the signal.
To detect cleaved Notch1 in metanephroi, the following modified method was used. After incubation of the primary antibody V1744 (1:500, Cell Signaling Technology, a division of New England Biolabs), the sections were treated with HRP-conjugated anti-rabbit IgG (1:1000; Jackson ImmunoResearch), tyramide-conjugated FITC (NEN, PerkinElmer; 1:1000), and then HRP-conjugated anti-fluorescein antibody. The antigen was then visualized with tyramide-conjugated Cy3 (Cy3 Plus, NEN; 1:400).
Detection of Lim1 was done with anti-Lim1 antibody (Chemicon International)
(Karavanov et al., 1996) with
slight modification to the manufacturer's protocol. The kidney was fixed in
MEMFA (MOPS 0.1 mol/l pH 7.4, EGTA 2 mmol/l, MgSO4 1 mmol/l, formaldehyde
3.7%) for 1 hour before embedding in paraffin. Rehydrated sections were boiled
in Trilogy (Cell Marque) for antigen retrieval, and incubated with anti-Lim1
antibody at 5 ng/ml (1:200 of stock). To visualize the antigen, it is
necessary to apply HRP-conjugated anti-rabbit IgG followed by
tyramide-conjugated Cy3. This allows double staining with other primary rabbit
antibodies like anti-Pax2.
For whole-mount staining, metanephroi were fixed in 4% PFA, washed in PBS and incubated in the blocking solution (MABT: 100 mmol/l maleic acid pH 7.5, 150 mmol/l NaCl, 0.1% Tween-20; plus 2% Blocking Reagent; Boehringer Mannheim) for one hour before adding anti-Ck8 antibody (1:10) for overnight incubation at room temperature. After extensive wash in MABT, the specimen was incubated with the Cy3-conjugated anti-rat IgG (1:1000). After this step, the metanephroi were incubated with FITC-LTL (1:200) for 1 hour at room temperature and then washed by PBS.
ß-gal staining to detect LacZ activity
The kidneys were fixed in 4% PFA for 2 hours before whole-mount ß-gal
staining at room temperature overnight. The specimens were embedded in
paraffin, sectioned and stained with antibody and/or counterstained with
diluted Hematoxylin or Nuclear Fast Red (Vector Laboratories). After
incubation with the primary antibody or FITC-conjugated LTL, the sections were
treated with HRP-conjugated IgG or anti-FITC antibody followed by color
development using diaminobenzidine tetrahydrochloride (DABT) as substrate.
Quantification of BrdU-labeling of cells
BrdU solution containing 5-Fluoro-2'-deoxyuridine (10% of the BrdU
concentration) was injected intraperitoneally in pregnant mice 2 to 3 hours
before kidney harvest. The samples were prepared and sectioned as described
above before being incubated with mouse anti-mouse BrdU antibody (1:200)
(Becton and Dickinson). It was visualized by Cy3 following HRP-conjugated IgG
incubation. The sections were then subjected to staining with another antibody
(Jag1 or Pax2) and then Hoechst nuclear stain. The single-color images were
merged into one RGB file magnified in Adobe Photoshop. For BrdU-labeled Pax2
cells, we counted the number of BrdU-stained Pax2-expressing cells and the
number of Pax2-expressing cells within the Pax2-expressing RVs or early
nephrons from one wild-type and one mutant kidney (19 RVs or early nephrons
from each sample; each sample contains multiple sections). The data were
presented as percentage of BrdU-positive Pax2 cells within the Pax2-expressing
cells. For BrdU labeling index of Jag1 cells, sections from three different
wild-type and three different mutant kidneys were included and 15 Jag1
clusters were counted in each kidney sample. Within each cluster we counted
the number of BrdU-stained Jag1-expressing cells and the number of
Jag1-expressing cells; and calculated the percentage of BrdU-labeled Jag1
cells in the Jag1-expressing cells. Student's t-test was used in the
first comparison, and one-way ANOVA in the second comparison.
P<0.01 was considered statistically significant.
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RESULTS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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), which
induces recombination in the metanephric mesenchyme
(Grieshammer et al., 2005;
Perantoni et al., 2005).
Kidneys from Pax3-cretg/+;
Rosa26Rtg/+ embryos
(Soriano, 1999) display LacZ
expression in all metanephric mesenchyme-derived tissues, including comma- and
S-shaped body and stromal tissue, while the UB derivatives remain unlabeled.
The early and broad recombinase activity assures an early and complete
recombination of alleles that is specific to the mesenchymal compartment and
their epithelial derivatives (see Fig. S1A,B in the supplementary material)
(see also Grieshammer et al.,
2005; Perantoni et al.,
2005). Viable, normal Pax3-cretg/+; N2f/f newborns were obtained at Mendelian ratios (data not shown). However, despite feeding successfully (data not shown), Pax3-cretg/+; N2f/f animals died 24 to 48 hours after birth. Gross anatomical examination revealed that Pax3-cretg/+; N2f/f had smaller kidneys than Pax3-cretg/+; N2f/+ siblings (Fig. 1A-D), and a small bladder suggested failure to produce urine (black arrows, Fig. 1A,C). During postnatal day 2 (P2), Notch2-deficient kidneys appeared to have lost vascular integrity (Fig. 1D). We observed hemorrhage into the interstitial spaces in Pax3-cretg/+; N2f/f P1 kidney (Fig. 1G, circle). The renal pelvis was collapsed, the papilla was flattened (see Fig. S2A,B in the supplementary material) and the collecting ducts (turquoise arrow, Fig. 1E-H) were less extensively branched than those of wild-type or heterozygote Pax3-cretg/+; N2f/+ littermates, even though N2f/f remains intact in the collecting duct network of the mutant. The nephrogenic zone, where the nephron initiating mesenchymal-to-epithelial transition takes place, appeared similar in thickness in both heterozygote and mutant kidneys (blue arrows, Fig. 1E,G), indicating a normal progression of the epithelialization process. By contrast, S-shaped bodies (yellow arrows), convoluted renal epithelia (green arrows) and glomeruli (red arrows), were not histologically distinguishable (Fig. 1E-H). Thus, the cause of death was renal failure due to the absence of a filtration apparatus. Heterozygote kidneys were indistinguishable from wild type in their morphological and histological features; hence we used `wild type' throughout to encompass both genotypes, although the exact genotype is detailed in the figures.
To address the state of nephrogenesis, we used immunohistochemical methods
to examine the residual renal tubules that were present in the mutant kidneys
(green arrowheads in Fig. 1G,H
and Fig. S2E in the supplementary material). Epithelial ductal labeling with
anti-cytokeratin 8 (Ck8; Krt8 - Mouse Genome Informatics) antibodies (specific
for UB derivatives) (Hemmi and Mori,
1991) confirmed that a branched collecting duct was present as
expected (Fig. 2C,E).
Expression of Wt1, a zinc-finger-containing transcription factor, expressed at
low levels in the metanephric mesenchyme (MM) and at high levels in podocyte
progenitors from the S-shaped body stage
(Fig. 2A), was detected only in
the MM surrounding the tips of the UB (Fig.
2B). Furthermore, whereas LTL, a marker specific for mature
proximal convoluted tubules (PCT), labeled numerous tubules in wild type at
E16.5 (Fig. 2D), no
LTL-positive structures were detectable in the mutants
(Fig. 2C). Thus, the
organization of proximal fates was clearly compromised by Notch2 removal. By
contrast, a comprehensive analysis of E-cadherin and Ck8 indicated that distal
nephrons were Notch2-independent. Both RV-derived epithelia and collecting
duct epithelium express E-cadherin, whereas Ck8 is expressed only in the
collecting ducts. Many E-cadherin-positive, Ck8-negative tubular structures
were detected in mutant kidneys. Several of these were continuous with the UB
tips (Fig. 2E,E').
Whereas proximal tubules also express E-cadherin, the lack of LTL-binding
activity, the continuity with the duct, their smaller size and the
regular-shaped lumen (see Fig.
1H and see Fig. S2E in the supplementary material) indicate that
renal tubules formed in the absence of Notch2 and most likely comprise only
distal tubule segments of the nephron. Together, these data suggest that
Notch2 is essential for the establishment of podocyte and PCT cell fates
during nephron segmentation.
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1-positive basal
lamina deposition (Abrahamson et al.,
1989) (Fig. 3C,D),
and the synthesis of epithelial adhesion molecules (E-cadherin,
Fig. 3E). Therefore, a robust
mesenchymal condensation and mesenchymal-epithelial transition occurs normally
in Notch2-deficient metanephric mesenchyme. The analysis of E-cadherin expression revealed three types of epithelial structures in the Notch2 mutant kidneys. First, the aforementioned early RVs: small cell clusters located just below the tips of the UB that expressed Pax2High (#1 in Fig. 3E); the second, larger epithelial clusters of Pax2 positive, E-cadherin-positive cells (#2 in Fig. 3E), a structure potentially analogous to comma- or S-shaped bodies or a `transitional' structure between the early RV and S-shaped body; and a third structure that was tubular in shape, Pax2Low and E-cadherin-positive (#3 in Fig. 3E).
We examined the expression of Wt1, cadherin 6 and E-cadherin for any
evidence of putative podocyte or proximal tubule precursors in the
Notch2-deficient renal epithelia. As was the case with LTL, cadherin 6, an
adhesion molecule thought to be expressed in the precursors of PCTs in
S-shaped bodies, was not detected in Notch2-deficient kidneys
(Fig. 4A,B). Further,
Wt1High podocyte precursors were also absent
(Fig. 4A',B'). On
close examination, the data suggested that proximal segmentation initiated,
but failed to establish independent proximal identities. During early stages
of nephrogenesis preceding formation of the S-shaped body, Pax2 is expressed
in all epithelia and is required to initiate expression of Wt1
(Dehbi et al., 1996);
upregulation of Wt1 inhibits Pax2 expression
(Dehbi et al., 1996;
Ryan et al., 1995). As in the
wild type (Fig.
4A',A''), we observed Wt1 expression in
Notch2-deficient nephrons that appeared to segregate from Pax2, such that
cells containing lower levels of Wt1 displayed relatively higher levels of
Pax2 and vice versa (Fig.
4B',B'';4A''',B'''). Further evidence of
polarity comes from analysis of Lim1 (Lhx1 - Mouse Genome Informatics); Lim1
expression is restricted to cells within the RV that are closest to the UB tip
(Fig. 4C), and this is observed
in Notch2 mutant kidney (Fig.
4D). Thus, the initiation of RV polarity appears to be
Notch2-independent, but the establishment of stable regional identities in
distinct proximal and distal regions of the developing nephron is defective
and proximal fates are absent from the S-shaped body.
To address Notch pathway activation in normal nephron segmentation, we
analyzed the spatial and temporal expression domain of Notch pathway proteins,
in conjunction with segment-specific markers. Jag1 protein was first detected
in clusters of Pax2Low-expressing epithelial cells in early RVs
(Fig. 4E). In the absence of a
good antibody to Dll1, we monitored the presence of LacZ knocked into the
Dll-1 gene in Dll-1LacZ heterozygotes
(Hrabe de Angelis et al.,
1997). Dll-1LacZ was also present in a subset
of RV cells (Fig. 4G) that also
contained activated Notch1 (Fig.
4H,I) and Lim1 (Fig.
4C). Jag1 expression and N1-ICD accumulation became elevated on
morphogenesis of the RV to the S-shaped body
(Fig. 3G,I,J,
Fig. 4C,G). Although the cell
fates have not been mapped to date, these observations suggest that activated
Notch1, Jag1 and Dll-1LacZ expression are likely to mark more
proximal precursor populations within RVs and their early derivatives and are
thus among the first markers of segmentation. Distal precursors within the
S-shaped body expressed E-cadherin and high levels of Pax2
(Pax2High) (Ryan et al.,
1995), but no Jag1 or N1-ICD
(Fig. 3F,H,I). Podocyte
precursors were Wt1-positive, Pax2Low- and Jag1-negative; some
contained N1-ICD (Fig. 3J,
Fig. 4F,I; see Fig. S3C in the
supplementary material). In the S-shaped body, Dll-1LacZ
expression overlapped with, but was broader than, the jag1 domain in
most S-shaped bodies. Whether this reflects real differences or the possible
perdurance of ß-galactosidase activity is unclear. Dll1LacZ
was detected in a few E-cadherin-expressing distal precursors and in some
podocyte precursors (see Fig. S4D in the supplementary material). In this
respect, the Dll-1LacZ expression domain resembled the
pattern of Notch1 activation better than the Jag1 domain (see Fig. S4C,D in
the supplementary material). We suggest that Jag1-positive,
Dll-1LacZ-expressing, N1-ICD-containing,
Pax2Low cells probably define proximal regional fates, and Jag1 is
likely to be a better marker of PCT precursors than Dll-1 (the
functional significance of Dll-1 is discussed below). Unfortunately,
while a detailed in situ expression analysis has been published for Notch
receptors (Chen and Al-Awqati,
2005; Leimeister et al.,
2003; Piscione et al.,
2004), the absence of a suitable antibody has prevented mapping of
Notch2 protein distribution.
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In conclusion, segregation of Wt1, Pax2 and Lim1 expression initiated and was accompanied by transient acquisition of a proximal precursor fate (Jag1-positive, N1-ICD present). However, podocyte precursors (Wt1High) never formed. This is because Notch2-deficient epithelial cells could not resolve proximal from distal fates, as evident from the simultaneous expression of markers typical for proximal and distal differentiation at the S-shaped stage. Notch2-deficient cells either die or adopt a Notch2-independent distal fate. We thus conclude that the transitional epithelial structures marked as #2 in Fig. 3E represent defective comma- or S-shaped bodies in which segmentation has initiated but the specification of appropriate regional identities has failed.
Notch2-deficient proximal precursors have reduced capacity to proliferate
Three hypotheses can explain why morphologically distinct comma- and
S-shaped bodies fail to form in the Notch2-deficient kidneys. First, cells
with dual identity (i.e. expressing both Jag1 and Pax2) die, resulting in
failure to form comma- and S-shaped bodies. Second, due to global
proliferation defects in the early renal epithelia, abnormal epithelial
structure forms. Third, Jag1-positive proximal tubule precursors are unable to
expand, and, in their absence, typical S-shaped bodies fail to form.
The first possibility was addressed by examining the distribution of active
caspase3, an early marker of apoptosis; no significantly enhanced apoptosis
was observed (data not shown). The second explanation was addressed by
pulse-labeling S-phase cells with BrdU and the fraction of Pax2High
cell examined in renal epithelia close to the UB tips; we observed a
BrdU-positive fraction of 
50% in both wild-type and Notch2
mutant metanephroi (54 versus 48%, P>0.1;
Fig. 5). Thus, a general
proliferative defect does not underlie abnormal tubule morphogenesis in Notch2
mutants. When proliferation was scored specifically in Jag1-expressing cells,
the subpopulation of Notch2-deficient, Pax2High,
Jag1-expressing cells entered the cell cycle twofold less frequently
than their wild-type counterparts (F(6, 15)=8.697,
P<0.001; Fig. 5).
Therefore, Notch2 activity is required for normal proliferation of proximal
regional precursors.
Notch1 is not required for cell fate determination during early nephron formation
To test if Notch2 was sufficient on its own for nephron segmentation in the
absence of any Notch1 input, we generated Pax3-cretg/+;
N1f/f embryos. However, these failed to survive beyond E9.5
(data not shown) and consequently were uninformative. Two alternative
strategies were adopted. First, we used chimera analysis with ES cells
deficient for Notch1 (Fig. 6).
Second, we used Pax2-cre transgenic mice
(Fig. 7)
(Ohyama and Groves, 2004).
We examined chimeric kidneys generated by injecting
N11/1;
Rosa26-lacZtg/+ ES cells (see
Hadland et al., 2004;
Nichols et al., 2004) into
wild-type blastocysts. N11 contains a large deletion of the
locus (Conlon et al., 1995). We
analyzed seven chimeric mice with Rosa26tg/+,
N11/1
cells and four with matched Rosa26tg/+ wild-type controls.
In all, lacZ-positive cells contributed extensively to normal-looking RVs,
S-shaped bodies and elongating nephrons, consistent with the absence of an
early function for Notch1 (Fig.
6B). Many glomeruli were composed entirely from
Notch1-deficient podocytes. The number of N11
podocytes that surrounded a wild-type capillary tuft were within the normal
range (Fig. 6F). Further, the
contribution of the Notch1-deficient cells to the LTL+
proximal convoluted tubules was also extensive
(Fig. 6D,D'). Thus, there
is no cell-autonomous requirement for Notch1 activity to establish proximal
renal tubule fates; however, we could not rule out a non-autonomous
contribution from Notch1-expressing cells intermingled with N1-/-
cells.
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). Compound heterozygote
Pax2-cretg;
N1f/1 embryos were
normal at E12.5 but died at E13.5 from unrelated vascular failure and
hemorrhage in the internal organs (data not shown). We therefore removed the
E12.5 metanephroi from Pax2-cretg/+;
N1f/1 embryos and
littermate controls (some of which also carried the
Dll1lacZ allele) and examined their development.
Notch1 protein is detected in the plasma membranes of duct and renal
epithelial cells (inset in Fig.
7C; see Fig. S3E-F in the supplementary material). Intense apical
staining indicates accumulation of Notch1 in S-shaped bodies (inset in
Fig. 7C; see Fig. S3E-F in the
supplementary material). Staining for Notch1 protein confirmed it was absent
in duct and renal epithelia of Pax2-cre containing metanephroi (inset
in Fig. 7D; see Fig. S3G-L in
the supplementary material). Accordingly, no accumulation of N1-ICD was
detected in renal epithelia of Pax2-cretg/+;
N1f/1 embryos (see Fig.
S3C-D in the supplementary material). By contrast to Notch2, Notch1-deficient
metanephroi appeared morphologically and histologically normal; they contained
LTL-positive proximal tubules (Fig.
7B), E-cadherin-positive and Ck8-negative distal tubules (not
shown) and Wt1High, synaptopodin-positive
(Mundel et al., 1997)
podocytes (Fig. 6D). By
contrast, Pax2-cretg/+; N2f/f embryos developed
kidneys lacking proximal tubule and podocytes (data not shown), indicating
that Pax2-cretg/+ used in this study removes Notch alleles
before the critical window described previously
(Cheng et al., 2003). Thus,
Notch1 is not required for regional organization of distinct cell fates.
Rbp-J deletion mimics the effect of Notch2 deficiency on nephron formation
One possible explanation for the crucial role of Notch2 and the failure of
active Notch1 signaling to normally complement Notch2 action would be that
Notch2 has acquired a unique, Rbp-J-independent activity. To address this
issue, we generated Pax2-cretg/+; Rbp-Jf/f mice
(Tanigaki et al., 2002).
Pax2-Cretg/+;Rbp-Jf/f embryos were normal at
E12.5 but died at E13.5; when cultured at E12.5, metanephroi from
Pax2-cretg/+; Rbp-Jf/f embryos branched
properly but failed to produce LTL-positive proximal convoluted tubules or
Wt1High podocytes (Fig.
7E-H). Thus, it appears that Notch2 acts in a conventional,
Rbp-J-dependent pathway during nephron segmentation.
Notch1 can stimulate proximal fates and inhibit distal ones when ectopically activated in nephron precursors
A second possible explanation for the failure of Notch1 signaling to
compensate for Notch2 could be a requirement for distinct levels of signaling
inputs; the existence of activation thresholds for the Notch targets Hes1 and
Hes5 was recently demonstrated in organ culture
(Ong et al., 2006). If so,
higher amounts of N1-ICD may be able to compensate for Notch2 in activating
its targets and promoting formation of proximal pattern.
|
|
). Hence, a
stable recombination results in a genetic modification of the MM and its
derivatives. To elevate N1-ICD levels, we created RosaNotch/+;
Six2-GFP::Cretg/+ mice. In these animals
Six2-GFP::Cre excises a `stop' cassette and constitutive expression
of N1-ICD activates Notch1 signaling
(Murtaugh et al., 2003). Pups
with this genotype were born in the correct Mendelian ratio but displayed
severely hypoplastic kidneys (Fig.
8A) in which the UB underwent a single branching event
(Fig. 8B). The absence of
Six2-producing cells at E13.5 in the RosaNotch/+;
Six2-GFP::Cretg/+ kidney suggested that lack of branching was
secondary to the loss of glial cell line-derived neurotrophic factor-producing
MM cells (data not shown). Despite the branching deficit, multiple tubular epithelial structures formed from the Wt1; Pax2 positive cell clusters (Fig. 8Ba-c). These tubular epithelia expressed both LTL (Fig. 8Bb,8C) and Slc34a1 (not shown), characteristic of PCTs. To determine whether activated Notch1 accelerated the formation of proximal tubules, E11.5 metanephroi were cultured for 4 days. At this stage, there were very few LTL-positive tubules present in wild type; however, metanephroi that overexpressed N1-ICD had already developed numerous LTL-positive tubules (Fig. 8C,C'). Furthermore, the activity of N1-ICD was independent of Notch2, as LTL-positive PCTs appeared even in the presence of DAPT (Fig. 9). In summary, N1-ICD can direct development of proximal nephron fates that are normally controlled by Notch2, consistent with a model in which N1-ICD is present at subthreshold levels during normal nephron patterning.
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). The
first epithelial structure (early RV stage) lacks distinctive morphological
features, although the expression patterns of genes such as Brn-1
(Nakai et al., 2003) suggest
that polarization starts soon after epithelialization. Within the asymmetrical
comma- and S-shaped body, molecular markers of the three main epithelial
lineages (distal tubules, proximal tubules and podocytes) map to discrete
epithelial domains. This process of renal epithelial segmentation may still
require `classical', UB-derived inductive function as mesenchyme induced for a
shorter time only gives rise to distal tubule fates, whereas the formation of
morphologically distinct proximal tubules and podocytes from MM requires at
least 24 hours exposure to the inducer
(Saxen, 1987). Moreover, the
ability of Pax2 to induce Wt1, and the ability of Wt1 to inhibit Pax2
(Ryan et al., 1995), should be
enough to support segregation of the podocyte lineage from the distal tubule,
but is not sufficient to account for formation of PCTs.
|
-secretase
activity (Cheng et al., 2003;
Wang et al., 2003), an enzyme
with many substrates, including Notch receptors
(Kopan and Ilagan, 2004).
Moreover, the sensitivity of renal epithelia to -secretase inhibitors
is informative: distal tubules are resistant to the drug, whereas proximal
tubules and podocyte are sensitive to it. Once S-shaped bodies form, they are
no longer sensitive to the inhibitor (Cheng
et al., 2003). This observation suggested that a
-secretase-dependent process acts to establish podocyte and proximal
tubule identities in RV derivatives. In this report, we identify Notch2 as the
first key determinant involved in acquisition of proximal fates in the
nephron.
The idea that Notch signaling may provide this function is supported by
marker analysis. Until recently, only a few markers had distinguished the
segments within the S-shaped body: Wt1 marks podocyte precursors
(Kreidberg et al., 1993),
cadherin 6 probable proximal tubule precursors, and E-cadherin and Brn-1 more
distal tubule precursors (Cho et al.,
1998; Nakai et al.,
2003). The usefulness of Pax2 as a segmentation marker has been
underappreciated; differential expression distinguishes prospective distal and
Bowman's capsule precursors from proximal precursors and podocytes (this
study) (Ryan et al., 1995).
Examination of all these markers, Notch ligands and Notch1 activation suggests
that segmentation initiates within the RV or its earliest derivative. Here, a
small subset of cells acquires proximal precursor markers (Jag1, activated
Notch1); expression may be activated at the boundary of Lim1-positive and
-negative territories. N1-ICD and Jag1 may provide the earliest markers for
proximal tubule precursors; we anticipate that this population expands and
expresses cadherin 6, and develops into mature proximal tubules that are
LTL+.
Our studies indicate that activated N1-ICD, Lim1 and Jag1 are all observed
in the RV of Notch2-deficient newborn mice; thus, it is likely that
segmentation initiates independently of Notch2. However, persistent expression
of the distal tubule marker (Pax2High) suggests a function for
Notch in Pax2 inhibition, while the reduced proliferation rates of
Jag1-expressing cells suggest Notch2 promotes the proliferative expansion of
this progenitor domain. Asymmetrical expression of
Dll-1LacZ in the RV is another early indicator of RV
segmentation (Fig. 4G).
Kobayashi et al. (Kobayashi et al.,
2005) have shown that Lim1-deficient kidneys produce lower
Dll-1 and arrest at the RV stage. Part of the Lim1 phenotype may be
explained by failure to activate Dll-1 and hence Notch2 at this
stage. Indeed, hypomorphic Dll-1 allele
(Kiernan et al., 2005) results
in reduction of proximal tubule formation (see Fig. S4 in the supplementary
material). These observations suggest that Lim1 can serve as an upstream
regulator of Notch ligands, and thus Notch signaling. However,
Lim1-/- ES cells do not contribute to the regions of the comma- and
S-shaped bodies, indicating that Dll1-expressing, wild-type cells cannot
rescue Lim1-deficient cells. Lim1 is thus required also during
proximalization. The early RV distribution of Lim1 was maintained in the
Notch2-deficient RV (Fig. 4D),
but in the more advanced nephron Lim1 expression assumed an abnormal pattern:
although they will all eventually adopt the distal fate, cells accumulating at
the proximal end expressed high Lim1 (Fig.
4C). Therefore, the separation of the distal and proximal lineages
and the differentiation of proximal tubule and podocyte precursors require two
parallel inputs, one provided by Notch2 and another from Lim1.
Notch1 and Notch2 have non-overlapping activities
We demonstrate here that while direct activation of Notch1 is observed in
proximal precursors of the S-shaped body, removal of Notch1 activity with
Pax2-Cre had no impact on the establishment of proximal fates. Thus,
Notch1 is clearly non-essential for this process and Notch2 is the only
-secretase substrate that plays a significant role in these patterning
events. As soon as Notch was identified as an X-linked locus in
Drosophila (Welshons,
1958), it was realized that the wing phenotype associated with
Notch mutations was due to haploinsufficiency. Notch1 is haploinsufficient in
vertebrates as well: myelination in the mouse is slowed in Notch1 heterozygote
animals (Givogri et al.,
2002), and human tricuspid heart valve development and maintenance
of valve flexibility throughout adult life require both alleles of Notch1
(Garg et al., 2005).
Importantly, no kidney disease is reported in these kindred; however, as this
manuscript underwent revisions, a human haploinsufficiency for Notch2 was
reported to cause Alagille syndrome
(McDaniell et al., 2006). This
would not be possible if Notch1 and Notch2 played redundant roles in human
nephron development; we infer from this data that in human, as in the mouse,
Notch2 is the dominant receptor during nephron segmentation.
While Notch1 and Notch2 differ in their ability to activate targets
(Ong et al., 2006), they have
the same affinity to Rbp-J (Lubman et al., 2006). Notch-responsive promoters
may respond differentially to similar nuclear concentrations of activated
Notch receptors within the nephron, becoming inactive if this amount falls
below a threshold (Ong et al.,
2006). What then can be the mechanistic basis for these
findings?
The observation that N1-ICD overexpression can promote the proximal fates
when endogenous Notch processing (and thus signaling) is abolished supports a
model whereby subthreshold levels of this protein in the normal renal
epithelium fail to complement Notch2 deficiency. Thus, Notch1 may be a weak
activator of key target(s) regulated normally by Notch2, or N1-ICD may fail to
accumulate to sufficient levels in the normal kidney to functionally replace
Notch2 deficiency. Another possibility is that N1-ICD is modified in a manner
that decreases its odds of association with Rbp-J. This will be predicted to
prevent its degradation (Fryer et al.,
2004), and ironically, facilitate its detection by
immunohistochemistry. When overexpressed, some N1-ICD may escape modification,
bind and activate crucial targets.
If differences existed between N1-ICD and N2-ICD that affected binding to
Rbp-J or to other putative partners, where would they map within the
intracellular domain? Domain swaps indicated that the divergent 426 amino
acids that lie downstream of the ANK domain are not important: mice expressing
a Notch2 hybrid containing this Notch1 domain (and thus deficient in the
Notch2 C-terminal domain) lack a kidney phenotype
(Kraman and McCright, 2005).
Crystallographic analysis of the Notch1 ANK domain revealed a surface unique
to each of the vertebrate Notch paralogs
(Lubman et al., 2005).
Therefore, the subtle differences in the ANK domain may be responsible for the
lack of redundancy between these highly conserved receptors (see also
Ong et al., 2006).
In conclusion, our study presents the first evidence for the crucial role
of Notch2 in an intrinsic patterning mechanism that establishes proximodistal
nephron polarity. We discovered a cell type in which two different Notch
molecules are present in its nucleus yet only one is crucial (Notch2), a
demonstration of unexpected complexity in this pathway, and provided evidence
that detection of N1-ICD per se is not an unambiguous indicator of a
functional role for Notch1 signaling. These results serve as an entry point to
explore further details of the mechanisms controlling early nephron
development. They establish that Notch2 acts to separate proximal and distal
fates, and in this Notch signaling most likely plays an instructive role after
an initial polarizing cue (Lim1?) acts in the RV. Thus, utilization of Notch
proteins in the kidney appears to be quite different from their use in the
skin, where all cellular identities emerge before, and independent of, Notch
function (Pan et al., 2004)
and where Notch1 plays a dominant role (S. Demehri, Y. Pan and R.K.,
unpublished).
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/4/801/DC1
|
ACKNOWLEDGMENTS |
|---|
|
REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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