1 Department of Molecular Biology and Pharmacology, Washington University School
of Medicine, Box 8103, 660 South Euclid Avenue, St Louis, MO 63110, USA
2 Department of Medicine, Renal Division, Washington University School of
Medicine, Box 8126, 660 South Euclid Avenue, St Louis, MO 63110, USA
3 Department of Physiology, The University of Texas Southwestern Medical Center
at Dallas, 5323 Harry Hines Blvd, Dallas, TX 75390, USA
4 Department of Pathology, The University of Alabama at Birmingham, 1530 3rd
Avenue, Birmingham, AL 35294-0019, USA
5 Department of Medicine, Division of Dermatology, Washington University School
of Medicine, Box 8103, 660 South Euclid Avenue, St Louis, MO 63110, USA
* Authors for correspondence (e-mail: kopan{at}molecool.wustl.edu and minerj{at}pcg.wustl.edu)
Accepted 1 July 2003
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SUMMARY |
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Key words: Metanephric culture, Notch, gamma-secretase, DAPT, Wt1, Mouse
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Introduction |
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Notch signaling plays a role in mammalian kidney development, but the
precise cellular steps that require Notch signaling are still obscure. The
permanent functional kidney, the metanephros, begins to form in the mouse at
11 days post coitum (dpc) when a collection of mesenchymal cells (the
metanephric blastema) induces an outgrowth from the Wolffian duct called the
ureteric bud, which invades the blastema
(Saxen and Sariola, 1987
). The
ureteric epithelium then induces adjacent mesenchymal cells to condense and to
epithelialize to form the renal vesicle
(Schedl and Hastie, 2000
).
Concomitantly, the mesenchymal derivatives induce branching and growth of the
ureteric bud. This process of reciprocal induction continues at the periphery
to form new nephrons and new ureteric bud branches in the mouse until 1-2
weeks after birth, resulting in
15,000 nephrons per kidney.
The renal vesicle differentiates and alters its morphology to form comma- and then S-shaped bodies, from which arise the nephrons that consist of glomerulus, proximal tubule and distal tubule. The ends of the distal tubules fuse with the inducing branches of the ureteric bud, which give rise to the collecting ducts. Each nephron segment expresses different proteins that serve as both cell fate and functional markers. The glomerular podocytes, together with endothelial cells, establish the glomerular filter between the blood stream and the urinary space.
McCright and colleagues (McCright et
al., 2001) have investigated the role of Notch2 in mammalian
kidney development. Notch2 mRNA was detected in comma-shaped bodies
and in tubules, including podocyte precursors. In addition, ureteric bud
branching and the mesenchyme-to-epithelial transition appeared normal in mice
homozygous for a hypomorphic Notch2 mutant allele. However, these
mice developed hypoplastic kidneys with glomerular defects. As the initial
formation of nephron precursors was normal in the mutant kidneys, Notch2 may
only be essential for glomerulogenesis. Alternatively, redundancy with other
Notch molecules may compensate for the deficiency in earlier Notch2 functions.
For example, Notch1 mRNA is detected in the renal epithelial
component of the mouse metanephros
(Weinmaster et al., 1991
;
Weinmaster et al., 1992
), and
Notch3 protein is present in the developing mouse kidney (data not shown). The
ligands Dll1 and Jag1 were detected in the comma- and S-shaped bodies, and
Jag1 expression persisted in the center of the developing glomerulus
(Beckers et al., 1999
;
McCright et al., 2001
).
Therefore, it is likely that the Notch signaling pathway plays a broad role in
mammalian kidney development.
Ligand binding to Notch triggers shedding of its extracellular domain by a
metalloprotease (Brou et al.,
2000; Lieber et al.,
2002
; Mumm et al.,
2000
). Subsequently,
-secretase-dependent proteolysis
within the transmembrane domain releases NICD
(De Strooper et al., 1999
;
Huppert et al., 2000
;
Schroeter et al., 1998
). All
four Notch molecules undergo intramembrane proteolysis
(Mizutani et al., 2001
;
Saxena et al., 2001
). To
investigate the role of Notch signaling in kidney development without the
problems associated with early embryonic lethality
(Gridley, 1997
) and functional
redundancy, we combined a pharmacological approach with metanephric organ
culture (Rogers et al., 1991
;
Saxen and Lehtonen, 1987
). By
culturing metanephroi in the presence of the
-secretase inhibitor DAPT
(Dovey et al., 2001
), we have
been able to block all
-secretase activity and therefore all
Notch signaling and record the consequences for kidney development.
Similar pharmacological studies with fetal thymus organ cultures
(Doerfler et al., 2001
;
Hadland et al., 2001
) or whole
animals (Geling et al., 2002
;
Micchelli et al., 2003
) were
extremely informative and demonstrated specificity for Notch signaling by
inhibition of
-secretase with no general toxicity. Moreover, by
transient application of DAPT at different time points, this method has
allowed us to define precise temporal dependence on
-secretase. These
studies have revealed an absolute requirement for
-secretase in the
transition from primitive epithelia to proximal nephron; missing are proximal
tubules and podocytes. Thus, Notch signaling appears to be involved in
patterning the proximal-distal axis of the nephron during
metanephrogenesis.
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Materials and methods |
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Ret activation assay
Ret-expressing Neuro2a cells stably transfected with GFR1-FLAG were
pre-treated (1 hour) with DMSO (1 µl/ml) or 1 µM DAPT in serum-free
media followed by stimulation with 40 ng/ml Gdnf in the presence of DMSO or
DAPT for the times indicated. Proteins were extracted for SDS-PAGE and blotted
as described (Tansey et al.,
2000
). Membranes were probed with rabbit polyclonal antibody
specific for phospho-p42/p44 (Thr202/Tyr204) from Cell Signaling Technology (a
division of New England Biolabs). Phospho-Ret PY905, PY1062 antibodies were
kindly provided by Drs Brian Tsui-Pierchala and Eugene Johnson
(Tsui-Pierchala et al.,
2002
).
Immunohistochemistry
The metanephroi or the embryos were fixed in Bouin's fixative or in 4%
paraformaldehyde (for DBA, LTL, or laminin 1 staining), embedded in
paraffin wax and sectioned at 7 µm. The sections were boiled in Trilogy
(Cell Marque) for antigen retrieval. The antibodies, the lectins and their
dilutions were as follows: WT1 (1:100, Santa Cruz), Pax2 (1:300; Covance),
Ncam (1:300, Sigma), E-cadherin (1:150; Transduction Labs), cytokeratin 8
(TROMA1; 1:10; Developmental Studies Hybridoma Bank), rat anti-mouse laminin
1 (1:1000; clone 8B3, kindly provided by Dale Abrahamson, Kansas City,
KS) (Abrahamson et al., 1989
),
rabbit anti-mouse cadherin 6 (1:300; kindly provided by Dr Dressler
(Cho et al., 1998
), Jag1
(1:150; Santa Cruz), and FITC-conjugated DBA and LTL (1:100; Vector
Laboratories). 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 were used to visualize the antigen in
Fig. 5K,L and
Fig. 8B, biotinylated
anti-mouse IgG and avidin-AMCA (vector) were used to detect E-cadherin.
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Quantification of branching morphogenesis
Focused, full-framed day 5 DBA whole-mount stained images were imported
into Adobe PhotoShop 5 for layout. To obtain the number of the branch tips,
the outermost branched tips pointing toward the margin of the specimen were
counted. Using the software's measure tool, the diameter of each marginal
branch tip at its widest was determined. The result was presented as
mean±s.e.m. for DMSO (n=61) and DAPT groups (n=29).
Tracing back from the tip to the main collecting duct, each end-branch could
be assigned as the result of a bifurcation number (one specimen in DMSO group,
n=29; two specimens in DAPT group, n=29). The result was
presented as the frequency distribution of the bifurcation number for both
groups. Student's t-test was used to compare the difference, and
P<0.01 was considered to be statistically significant.
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Results |
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Inhibition of -secretase with DAPT prevents Notch
activation
To study the role of Notch signaling in early mammalian kidney development,
we treated cultured mouse metanephroi with DAPT in order to inhibit
-secretase activity and therefore block all Notch signaling. As
controls, we cultured the contralateral kidney in the presence of the solvent
used, dimethyl sulfoxide (DMSO). NICD immunoreactivity
(Fig. 1B) disappeared when DAPT
was present (Fig. 1F). This
demonstrates that DAPT penetration of cultured metanephroi was sufficient to
completely block Notch proteolysis.
DAPT attenuates branching morphogenesis without affecting the
Gdnf-Ret pathway
After 3 days in DAPT, fewer epithelial structures were present compared to
the untreated contralateral side, and this became more obvious after 5 days
(compare Fig. 1C,D with 1G-H).
The missing epithelial structures could result from defects in ureteric bud
branching, mesenchyme-to-epithelium transition, epithelial growth and
differentiation, or a combination of these. To analyze the effects of
-secretase inhibition on branching morphogenesis, we used
fluorescein-conjugated Dolichos biflorus agglutinin (DBA), which
labels the ureteric buds and their derivatives, collecting ducts
(Laitinen et al., 1987
). After
2 days of culture, there was no discernible difference in branching. However,
after 3 days, the treated metanephroi had reduced arborization and more
dilated end-branches (Fig. 2).
We used three parameters to quantify the changes in several 5-day-old
specimens: the number of bifurcations, the number of branch tips at the
periphery and the average diameter of branch tips (see Materials and methods).
The distribution of branch bifurcations between the two growth conditions was
significantly different (P<0.01): control metanephroi had
10
on average, and no branches had fewer than six branch points (9.8±0.3,
n=29); DAPT-treated ones had at most eight branch points
(7.1±0.3, n=29; Fig.
3A). As a consequence, controls contained twice as many branch
tips at the periphery (32 or 29 in DMSO versus 14 or 15 in DAPT). Control
branch tips appeared finer (46.3±1.6 µm, n=61, versus
80.0±3.3 µm, n=29; P<0.01) with a constant
diameter. By contrast, the end-branches of DAPT-treated metanephroi were
larger and irregular in diameter and had longer segments lacking ramification
compared to untreated metanephroi. Thus, DAPT retarded branching. In addition,
small cone-shaped protrusions emerged out of branch shafts (not shown), and
the epithelium at the tip of a few end branches pinched to form bubble-like
structures (Fig. 2, inset).
These may represent incomplete or aborted branching or ectopic branch
points.
|
|
DAPT diminishes, but does not completely abolish, the
mesenchymal-epithelial transition
Hematoxylin and Eosin stained sections of metanephroi cultured for 5 days
revealed fewer renal tubular epithelia and more interstitial cells in the
DAPT-treated metanephroi (Fig.
4), consistent with the overall morphology
(Fig. 1). Few, if any,
comma-shaped and S-shaped bodies or glomeruli were visible in DAPT-treated
samples. A slight increase in activated caspase 3 staining was observed at the
periphery of DAPT-treated metanephroi, consistent with the possibility that
cell death was increased (data not shown). As seen in the DBA whole-mount
stains, dilated collecting ducts were found in the DAPT-treated metanephroi
(asterisks in Fig. 4E,G). To
identify epithelial cells accurately, we analyzed the expression of several
epithelial cell markers. Neuronal cell adhesion molecule (Ncam, green) is
expressed in uninduced and condensed mesenchyme and in comma and S-shaped
bodies, and then it is lost (Klein et al.,
1988; Nouwen et al.,
1993
). The same sections were stained for Pax2 (red in
Fig. 4B,F), a paired
domain-containing transcription factor expressed in the condensing mesenchyme,
the newly formed renal epithelia and the collecting ducts, but not in the
uninduced mesenchyme or in the podocytes
(Dressler et al., 1990
;
Patterson and Dressler, 1994
).
Pax2 staining was conducted on adjacent section of most panels (not shown).
Ducts (Pax2 positive, Ncam negative; Fig.
4B,F, arrowheads) in both control and treated metanephroi were
surrounded by condensing mesenchymal cells positive for both Ncam and Pax2.
Strikingly, fewer organized epithelial structures lacking Pax2 but expressing
Ncam (Fig. 4B, arrow, inset)
were detected in DAPT-treated metanephroi compared with control. Instead,
increased abundance of Pax2-negative, Ncam-positive cells were present in
DAPT-treated metanephroi (Fig.
4F, asterisks). Ncam-positive cells closer to the tips of the
ureteric bud were likely to have received inductive signals, and therefore
proceeded to express the marker Pax2, identifying them as condensing
mesenchyme. However, many of these cells failed to epithelialize
(Fig. 4E,F). This indicates
that
-secretase inhibition did not block mesenchymal condensation, but
attenuated the transition to epithelium.
|
-Secretase activity is required for the differentiation of the
proximal epithelial derivatives
Next, we sought to determine to what extent -secretase inhibition
prevented differentiation of primitive renal epithelia into mature nephron
segments. To identify additional differentiated epithelial cells, we first
examined the expression of the Wilms' tumor protein Wt1, a zinc-finger
transcription factor that is expressed at a low level in uninduced and induced
metanephric mesenchyme, in condensing mesenchyme, and in early epithelia, but
is strongly upregulated in glomerular podocytes
(Schedl and Hastie, 2000
).
Metanephroi were first examined for global up-regulation of Wt1 expression by
whole-mount antibody staining (Fig.
2). After 1 day in culture, the expression of Wt1 was mainly
detected in the region surrounding the bud tips
(Fig. 2). At that time point,
DAPT-treated metanephroi showed a similar expression domain at comparable
levels. After 3 days in culture, elevated Wt1 expression appeared in podocyte
clusters within developing glomeruli (arrows,
Fig. 2). As with branching,
differences in podocyte/glomerulus formation became obvious with every
successive day in culture: in the control samples, numerous intensely positive
Wt1 (Wt1HIGH) glomeruli could be seen
(Fig. 2,
Fig. 7A), but few such
structures were found in the treated samples. Their numbers did not increase
after day 3, and they were always located near the center
(Fig. 2,
Fig. 7B). In both the treated
and untreated samples, weak Wt1 expression (Wt1LOW) could be
detected in the region surrounding the collecting ducts each day of culture
(Fig. 2). We further examined
Wt1 expression in cross-sections of cultured metanephroi. In the control, the
Wt1HIGH podocytes formed aggregates throughout the tissue,
including the periphery (Fig.
5A-B, Fig. 7E). In
DAPT-treated metanephroi, only few podocytes and well-defined glomeruli
formed, primarily in the center (Fig.
5E-F, Fig. 7F).
This is in contrast to E-cadherin-positive, cytokeratin 8-negative
(mesenchyme-derived) epithelia that formed in DAPT-treated metanephroi
throughout the section, including the periphery. This result implies that new
renal epithelia are still forming at the periphery even in the presence of
DAPT (Fig. 5B,F, asterisk,
podocyte overlay in blue, see below), but they are unable to give rise to
podocytes.
|
Finally, we analyzed the E-cadherin-positive, cytokeratin 8-negative (Figs
4,
5) epithelial structures we
classified as distal tubules. We postulated these cells were derived from a
population of Wt1LOW/Pax2-positive cells at the periphery of
DAPT-treated metanephroi (Pax2 staining not shown). However, the reduced
numbers of these structures could indicate that a general block in
epithelialization is imposed during DAPT treatment, and that the observed
distal structures formed from renal epithelial cells pre-existing in the E12.5
metanephroi. If epithelia were able to form from mesenchyme in the presence of
DAPT, we reasoned that they should express both E-cadherin and Pax2 shortly
after epithelialization, while Pax2 would be downregulated as the epithelium
matured (Klein et al., 1988;
Patterson and Dressler, 1994
).
Indeed, we detected Pax2 in the nuclei of E-cadherin-positive, cytokeratin
8-negative epithelia (inset in Fig.
5L, periphery on the left), suggesting that they were newly formed
from conversion of mesenchyme. A more definitive approach to clarify the
origin of epithelia in the presence of DAPT was to harvest the metanephroi
from E11.5 mouse embryos, before any renal epithelia are formed. The first
T-shaped bifurcation of the ureteric bud formed after 1 day in culture (data
not shown), indicating that renal epithelial cells could not have existed
prior to tissue collection. After 6 days of culture with DAPT,
E-cadherin-positive, cytokeratin 8-negative epithelia formed, demonstrating
that mesenchymal-epithelial transition indeed occurs in the absence of
-secretase activity (Fig.
5L). Similar to the experiment with E12.5 metanephroi,
Wt1HIGH podocytes appeared only in the DMSO-treated control
(Fig. 5K).
-Secretase activity is not essential for podocyte
differentiation
In the section above we established that distal tubules form but proximal
structures (podocytes, proximal tubules) are completely missing when DAPT is
present during the induction process. However, in addition to this early
requirement for -secretase activity, the defects in mice expressing a
hypomorphic allele of Notch2 suggest that later differentiation events may
also require this enzyme (McCright et al.,
2001
). To determine whether Notch signaling is directly involved
in podocyte differentiation, we inhibited
-secretase in older
metanephroi, isolated at 14.5 dpc. At this age, the center of the tissue
contains glomeruli, and less mature cells are present peripherally
[Fig. 6A, from the center
outwards: D, podocytes (Wt1HIGH); C, comma and S-shaped bodies
(Pax2, Ncam, E-cadherin, NICD, Jag1); B, condensed mesenchyme (Pax2, Ncam,
Wt1LOW); A, mesenchymal stem cells (Ncam, Wt1LOW]. If
only induction of proximal structures requires
-secretase, the many
S-shaped bodies that are present at 14.5 dpc should be able to form new
glomeruli at peripheral positions in both treated and untreated metanephroi
after 3 days in culture (the time it takes for Wt1HIGH cells to
appear in 12.5 dpc metanephroi; Fig.
2). Only after 4 days in culture, a period of time sufficient to
generate glomeruli from mesenchyme, should there be a difference (model 1,
Fig. 6A). However, if Notch
signaling is required not only for the induction of proximal structures but
also for differentiation of proximal tubule cells and podocytes, new glomeruli
will not be generated from pre-existing S-shaped bodies in DAPT-treated
cultures. Hence, the Wt1HIGH glomeruli are expected to mainly exist
in the center of the developing kidney at the 3-day point (model 2 in
Fig. 6A). Consistent with Model
1, 3-day culture of 14.5 dpc metanephroi results in similar distributions of
Wt1HIGH glomeruli throughout the organs in both control and
DAPT-treated tissues (Fig. 6B).
At day 4, the number of glomeruli at the periphery was markedly decreased in
DAPT-treated metanephroi compared with the controls, and the glomeruli were
now exclusively found in the center of the sections
(Fig. 6C). These findings
suggest that
-secretase inhibition blocks the formation of proximal
structures but not their differentiation once S-shaped epithelia form.
|
To confirm that -secretase activity was restored, we assayed for
activated Notch1, as above. Twelve hours and 36 hours after DAPT removal, no
NICD was detected (Fig. 7K and
data not shown); however, NICD immunoreactivity returned by 60 hours
(Fig. 7L). We could also detect
a few comma- or S-shaped bodies that formed within 60 hours, and persisted
after 84 hours following DAPT removal (NICD/Jag1-positive,
Fig. 7J,L,M). This observation
prompted us to investigate whether any proximal epithelial differentiation
occurred. We therefore examined the expression of laminin
1 and
E-cadherin. There were many laminin
1 and E-cadherinLow
double-stained epithelial structures after 3 or 4 days recovery following 3.5
days of DAPT treatment (Fig.
8A,B). These NICD/Jag1 structures could be newly formed proximal
renal epithelia as they started to synthesize basement membrane, and showed a
low level of E-cadherin. Indeed, cadherin 6-positive proximal tubules were
detected in the same metanephros after 2.5 days without DAPT
(Fig. 8C). We confirmed that
NICD-expressing epithelia were also cadherin 6-positive (data not shown).
Thus, after 2 days of DAPT treatment, cells retained the ability to choose all
proximal fates, but after 3.5 days of DAPT treatment, cells lost the ability
to form podocytes while retaining the ability to form proximal tubules.
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Discussion |
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Pharmacological inhibition of Notch activation in cultured mouse
metanephroi attenuates branching and reveals a crucial role in formation of
proximal epithelial structures
We find that ureteric bud branching proceeded quite extensively even in the
absence of -secretase activity. The difference in branching between the
control and the treated kidneys was statistically significant but not
dramatic. As
-secretase also cleaves other Type I transmembrane
proteins including Erbb4, a receptor tyrosine kinase
(Lee et al., 2001
;
Ni et al., 2001
), we examined
the possibility that DAPT might directly affect the Gdnf-Ret pathway, a major
regulator of the ureteric branching mediated through the receptor tyrosine
kinase. Our data did not support such an effect. Collectively, our results
indicate that although branching persists at a reduced rate, the inhibition of
-secretase interrupts the tightly regulated association between growth
and ramification (Davies and Davey,
1999
), leading to an increase in shaft diameter without
bifurcation. One possibility is that the branch-inducing cells are diminished
in the treated metanephroi, or that their inductive potential is altered.
Another possibility is that unknown
-secretase substrate(s) in the
ductal cells are directly affected.
Mature nephrons form in two steps: first, mesenchymal-epithelial transition
(MET) is induced by the ureteric bud; second, segmental differentiation of the
renal epithelium occurs to form the podocyte, the proximal tubule and the
distal tubule, in that order (Saxen,
1987). Given that a general reduction in the number of renal
epithelial cells (fewer E-cadherin-positive structures) was observed in all
DAPT treated metanephroi, we considered the following four possibilities: (1)
-secretase inhibition blocks MET; (2) cellular differentiation still
occurs, but proper epithelialization fails; (3) inhibition of formation of the
earlier cell type (podocytes) precludes differentiation of later cell types
(proximal and distal epithelia); or (4)
-secretase inhibition
interrupts only the differentiation of a particular epithelial cell type. Our
data using 11.5 dpc metanephroi, which did exhibit MET in the presence of DAPT
(Fig. 5), argue against the
first scenario. However, MET was significantly attenuated, and this may be due
to the presence of unknown MET-promoting
-secretase substrates in
mesenchymal cells the activities of which are blocked by DAPT. In the second
scenario, the few epithelial structures (or non-epithelial cells surrounding
the ducts) should express differentiation markers of all cell types, but they
do not. In the third scenario, all segment-specific markers will be lost, but
they are not, as E-cadherinHIGH distal tubules are present. In the
last scenario, only some cell types would be lost, and that is what we
found.
Interestingly, once formed, podocyte precursors were able to differentiate
to WT1HIGH cells even in the presence of DAPT, and this may seem
inconsistent with the phenotype of mice homozygous for a hypomorphic
Notch2 allele (McCright et al.,
2001). The Notch2-sensitive step discovered by McCright et al.
during glomerulogenesis may require the vasculature or is reached at a stage
not recapitulated in organ cultures. Notch3 is expressed in the podocytes, but
Notch3-null mice are apparently normal, suggesting no or a minimal
role in glomerular formation and function. A model for the role of Notch
signaling in early metanephros development is presented in
Fig. 6.
Prolonged inhibition of the -secretase activity may eliminate
the differentiation potential of the renal epithelial cells
From our recovery experiments, we conclude that all epithelial cell types,
including podocytes, can form after 2 days without -secretase activity
(inset in Fig. 7I). If devoid
of
-secretase activity for more than 3 days, renal epithelial cells
irreversibly lose their ability to form podocytes. Instead NICD-containing
epithelial structures express low levels of E-cadherin, laminin
1 and
cadherin 6, indicating that proximal tubules formed in the absence of
podocytes (Fig. 8 and data not
shown). Furthermore, the correlation between the resumption of branching and
Notch activation suggests that NICD-positive epithelial cells contribute
indirectly to branching.
Our finding that all cell types can form after 2 days inhibition, proximal
and distal tubules form after 3 days inhibition, and only distal tubules form
under continuous inhibition is intriguing. Based on the experiment in which
mesenchyme was induced by exogenous spinal cord signals, Saxen
(Saxen, 1987) suggested that
the differentiation of a nephron is segmental and sequential
(podocytes
proximal tubule
distal tubules). This suggested sequence
was deduced from the observation that short induction time (<24 hours)
produced predominantly glomeruli and proximal tubule but no detectable distal
tubules. Our results demonstrate the inverse order of sensitivity to
-secretase inhibition;
-secretase activity, probably through
Notch1, is required specifically for the proper induction of the proximal
epithelial structures (but is not needed for the differentiation of podocytes
once they have been induced, Fig.
6). We therefore can conclude that each segment is able to
differentiate without signals from the cells proximal to it. Although
differentiation of more proximal cells is not a prerequisite for the
subsequent differentiation of the distal structures, it may still be required
for maximal success in epithelialization in our experiments.
A related finding was reported earlier regarding the role of Notch in cell
fate determination in vertebrate pronephros development
(McLaughlin et al., 2000). In
Xenopus, Notch1 mRNA was first detectable in the developing
pronephrotic anlage during early tail bud stages. During late tail bud stages,
Notch1 expression became restricted in the epithelial pronephric
tubules. When Notch signaling was blocked by injecting the embryos with RNA
encoding a dominant-negative CSL, the tubular cell fate was converted to the
ductal fate. Notch is thus essential for selecting the type of renal
epithelial cell both in the Xenopus pronephros and in the mouse
metanephros.
Is Notch1 participating in lateral interaction within the metanephros?
Overlap of Notch expression with its ligands has been noted in the kidney
(Leimeister et al., 1999). The
precise cellular colocalization of activated Notch1 with its ligand, Jag1
(Fig. 7J, inset, enlarged image
in supplemental Fig. S1 at
http://dev.biologists.org/supplemental/),
and their uniform intensity is inconsistent with the salt-and-pepper pattern
of NICD accumulation anticipated from a simple lateral inhibitory role for
Notch signaling within the renal epithelium
(Artavanis-Tsakonas et al.,
1995
; Artavanis-Tsakonas et
al., 1999
; Bray,
1998
). In addition, Jag1 expression may depend on
-secretase and therefore on Notch; it reappears with NICD when DAPT is
removed (Fig. 7K-M; H. Zheng
and P. Wang have noted this as well, personal communication). This, too, is
contrary to the predicted behavior of Notch ligand in laterally interacting
cells (Wilkinson et al.,
1994
). Determination of the precise role of Notch1 in kidney
development, whether the Wt1LOW/Pax2-positive cells acquired novel
properties or are simply expanding nephron progenitors, and how Jag1
expression is regulated are beyond the scope of this report.
Finally, our results have significant implications for treatment of
Alzheimer's disease. As the presenilin-dependent -secretase processes
the amyloid precursor peptide to generate fragments that are causally related
to Alzheimer disease,
-secretase is currently an important therapeutic
target (Dovey et al., 2001
).
Although individuals with Alzheimer's disease need not worry about any drug
effects on fetal development, the kidney is a major collateral target organ of
disease processes and drug toxicity in the elderly. Preservation of its
limited regenerative capacity is a very important clinical concern. Imgrund et
al. (Imgrund et al., 1999
)
showed that Pax2, which is not normally expressed in adult tubules,
was expressed in the regenerating proximal tubular epithelium following
tubular necrosis induced by folic acid, implying that the kidney may
recapitulate its developmental process to regenerate. If the role of
-secretase (and Notch) during kidney regeneration is similar to the
early developmental process,
-secretase inhibitors may prevent an
injured kidney from regenerating properly.
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ACKNOWLEDGMENTS |
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Footnotes |
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REFERENCES |
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