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FIG. 1. Temporal expression of Notch-1, Delta-1, and Serrate-1 during pronephric development. (A) Schematic of a nonintegrated nephron.
Wastes are filtered from the glomus into the coelom where fluids are swept into the kidney tubules by the thin ciliated funnels
(nephrostomes). Any molecules not resorbed in the pronephric tubules are disposed of via the pronephric duct. (B) Expression patterns of
Delta-1, Serrate-1, and Notch-1 via whole-mount in situ hybridization between stages 21 and 34. Embryos are shown laterally with anterior
to the right and dorsal to the top. The pronephric anlage is located just posterior to the head and ventral to the anterior somites. Note the
dynamic expression patterns of both ligands within the pronephric anlage, as the expression pattern of Notch pathway components becomes refined during kidney morphogenesis. Red arrows indicate the region in which the pronephros is located, but no Delta-1 (d) or
Serrate-1 (e) expression can be detected at these stages. Black arrows mark the position of Delta-1, Serrate-1, or Notch-1 expression in the
pronephros. (C) Time-line summary of Delta-1, Serrate-1, and Notch-1 expression patterns determined by whole-mount in situ
hybridization. Note that Delta-1 expression is observed in the pronephric anlage (stage 19) prior to detection of Serrate-1 expression (stages
22 23). Unlike Serrate-1 expression, which continues to be expressed until tadpole stages, Delta-1 expression decreases and is no longer
detectable by stages 33 34. The expression of the receptor, Notch-1, is detected from the onset of kidney organogenesis (early tail bud) and
continues to be expressed until the pronephric kidneys are fully functional (stages 38 39).
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FIG. 2. Notch signaling regulates endogenous ligand expression. The expression of Delta-1 and Serrate-1 was examined in embryos injected with
mRNAs encoding either GR-Su(H)DBM (dominant negative) or GR-Su(H)VP16 (activated) into one ventrovegetal blastomere at the eight-cell stage.
As a lineage tracer, mRNA encoding b-galactosidase was co-injected and visualized using the substrate magenta-gal. At stages 19â20 constructs
were induced via the addition of the glucocorticoid dexamethasone to the injected embryos. Embryos were cultured until fixation at the desired
stage and examined by whole-mount in situ hybridization. (A) Lateral views of Delta-1 or Serrate-1 expression on the uninjected side of an embryo
(a, c) compared to the GR-Su(H)VP16-injected side (b, d). Note the decrease of Delta-1 expression on the injected side of the embryo when the Notch
pathway is activated in the region of the developing pronephros [black arrow in (b) marks the position of normal Delta-1 expression]. In contrast,
the expression of Serrate-1 is substantially increased on the GR-Su(H)VP16-injected side of the embryo [yellow arrow in (d) indicates region where
Serrate-1 expression is increased in the tubule anlage; red arrow in (d) identifies the expansion of Serrate-1 expression into a more posterior region
of the pronephros that normally forms the pronephric duct]. (B) The top two images show lateral views of Delta-1 expression on the uninjected (a)
and the GR-Su(H)DBM-injected (b) sides of an embryo. Note that Delta-1 expression is elevated on the injected side [black arrow in (b)], whereas
Serrate-1 expression is diminished [black arrow in (d) indicates the normal position of Serrate-1 expression].
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FIG. 3. Activation and suppression of Notch signaling has opposite effects on pronephric duct formation. Opposite effects on GR-Su(H)VP16-
(activated) and GR-Su(H)DBM- (dominant negative) injected embryos were observed when examining markers of the pronephric duct by either in situ
hybridization or antibody staining using markers of various stages of duct differentiation (c-ret, Lim-1, 4A6 epitope). Injected constructs were
induced at stages 19â20 by the addition of dexamethasone. Note that injection of GR-Su(H)VP16 resulted in almost complete obliteration of all
three molecular markers of duct morphogenesis on the injected side of the embryos (B, F, J) compared to the uninjected control side (A, E, I). An
opposite effect was detected in GR-Su(H)DBM-injected embryos, in which an increase of duct markers was observed on the injected side of the
embryos (D, H, L). Black arrow in (J) indicates the location of the missing Lim-1 expression in the pronephric duct. The black arrow in (L) marks
the increased expression of Lim-1 in the anterior portion of the injected embryo. Note that the three patches of Lim-1 expression which presumably
mark the nephrostomes [yellow arrowheads in (K)] are less distinct after expression of GR-Su(H)DBM [red arrow in (L)].
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FIG. 4. Perturbations in Notch signaling cause defects in pronephric
duct morphology. GR-Su(H)VP16- (activated) or GRSu(
H)DBM- (dominant negative) injected embryos were examined by
either whole-mount in situ hybridization or antibody staining
using the following markers of duct formation: Lim-1, c-ret, and
4A6 epitope. As a control, injected sibling embryos were reared in
the absence of dexamethasone, which resulted in minimal effects
on duct marker expression (no dexamethasone GR-Su(H)DBMinjected
embryos Lim-1 16% n 5 103, 4A6 10%n 5 69, c-ret 16%
n 5 63; no dexamethasone GR-Su(H)VP16-injected embryos Lim-1
15% n 5 459, 4A6 18% n 5 83, c-ret 15% n 5 82). Injected,
dexamethasone-treated embryos resulted in dramatic effects on
duct marker expression on the injected side of the embryo compared
to the uninjected side (dexamethasone-treated GR-Su(H)DBM
embryos Lim-1 69% n 5 174, 4A6 72% n 5 148, c-ret 53% n 5
103; dexamethasone-treated GR-Su(H)VP16 embryos Lim-1 77%
n 5 408, 4A6 88% n 5 213, c-ret 73% n 5 261). Fewer than 10%
of uninjected dexamethasone-treated embryos showed asymmetry
of all markers examined comparing the left and right sides of the
embryo (data not shown). A change (either an increase or decrease)
of molecular markers of the pronephros was determined by visual
inspection of the injected side of the embryo compared to the
uninjected control side of the embryo by two independent investigators.
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FIG. 5. Notch signaling affects expression of markers of the tubules and glomus. Markers of either tubules (Pax-2 or 3G8 epitope) or the
glomus (Wt-1) were used to examine the affects of activation or suppression of Notch signaling on kidney organogenesis. (A) The right-hand
column shows lateral views of GR-Su(H)VP16- (activated) injected embryos. The left-hand column shows the expression on the uninjected side of embryos. The in situ probe Pax-2 and the 3G8 antibody were used to examine tubule organization and morphogenesis. Pax-2
expression did not show a normal tubule pattern, but instead was expanded in the tubule anlage on the injected side of the embryo.
Similarly, the injected side of the embryo displayed disorganized 3G8 monoclonal staining in the tubule anlage compared to the control
uninjected side. The developing glomus was examined using the molecular marker Wt-1. The expression of Wt-1 was dramatically
increased on the GR-Su(H)VP16-injected side of the embryo and in particular was expanded into a more posterior region. (B) A lateral view
of a 3G8 (tubule) antibody stained, GR-Su(H)DBM- (dominant negative) injected embryo. Suppression of the Notch pathway results in normal
tubule morphology (blue arrow). It is important to note, however, that extra b-galactosidase-positive cells (magenta-gal pink cells and black
arrow) were able to contribute to the region of the developing duct.
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FIG. 6. Signaling through NotchICD affects expression of markers of the duct, tubules, and glomus. Similar to the effects observed with
the GR-Su(H)VP16, activation of Notch signaling using an inducible NotchICD decreased molecular markers of the pronephric duct (Lim-1,
ret-1) and increased markers of the glomus (Wt-1) and the pronephric tubules (Pax-2) on the injected side of embryos in a dexamethasonedependent
manner. Blue arrows indicate the position of the missing pronephric duct markers. Black arrows indicate the increased
expression of either the glomal or the tubule marker indicated.
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