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The tumor suppressor WT1 has been demonstrated to have a wide variety of activities in vitro and is required for metanephric development in vivo. In the experiments presented here, the Xenopus pronephros was used as a simple model system to examine the activity of Xenopus WT1 (xWT1) during kidney development. xWT1 was ectopically expressed in Xenopus embryos by mRNA injection and found to inhibit pronephric tubule development. Confocal microscopy confirmed this observation and revealed that the inhibition was the result of a failure to form a pronephric anlage of appropriate size rather than a defect in epithelialization. Examination of Xlim-1 expression, an early molecular marker of pronephric specification, in tailbud embryos indicated that injected xWT1 mRNA inhibited pronephric specification prior to any overt sign of morphogenesis (Xenopus stage 21). These results suggest that xWT1 may act to repress tubule-specific gene expression in the portion of the pronephros fated to form its vascular structure, the glomus.
FIG. 1. The pronephric index. The index quantifies the extent of pronephric development by comparing the left and right pronephroi
within an embryo (based upon the pattern of antibody 3G8 staining at stage 37/38). (A and B) Opposite sides of an embryo injected on one
side with 1 ng of b-galactosidase mRNA and stained with antibody 3G8 to detect pronephric tubules (arrow in A indicates staining).
Staining for b-galactosidase activity (light blue/green in B) indicates that injection into the C3 and D3 blastomeres has targeted mRNA to
the pronephric region of the injected side (B). (a9 and b9). Enlarged, diagrammatic representation of the pronephroi in A and B. For
quantification of pronephric development, each pronephros in an embryo is scored by assigning one point for each of the numbered tubule
components in the figure (a9 and b9). A normal Xenopus pronephros has five components: one vertical tube, one horizontal tube, and three
attached branches (see Vize et al., 1995), so a score of 5 is assigned to a normal pronephros; 0 indicates no pronephros. The pronephric index
(PNI) is then expressed as the difference between the two sides. PNI 5 0 indicates two identical pronephroi; PNI 5 5 indicates a normal
pronephros on one side and no pronephros on the other. The embryo shown in A and B has a pronephric index of 0, indicating that injection
of b-galactosidase mRNA has no effect on pronephric development.
FIG. 2. Injection of xWT1-KTS mRNA inhibits the development of the pronephric tubules. Antibody 3G8 was used to detect the
pronephric tubules (dark purple in all panels), and X-Gal was used to detect the b-galactosidase (light blue/green in B and D). (A and B)
Opposite sides of an experimental embryo injected with 1 ng of xWT1-KTS mRNA (10.25 ng of b-galactosidase mRNA). The uninjected
side (A) is normal, while all distal branches of the collecting tubule are absent on the injected side (B). The diameter and length of the
remaining common tubule and collecting tubule are also reduced on the injected side (B) compared to the uninjected side (A). The control,
uninjected pronephros (diagrammed in a9) is normal and is assigned 5 points; the pronephros on the injected side of the same embryo
(diagrammed in b9) is lacking all distal branches of the connecting tubule and is assigned only 2 points. The resulting PNI for this embryo
is 3, indicating a significant inhibition of pronephric development (see Table 1). (C and D) Opposite sides of a different embryo injected with
1 ng of xWT1-KTS mRNA (10.25 ng of b-galactosidase mRNA) displaying a more severe phenotype. The uninjected pronephros (C) is
normal and is assigned 5 points (c9). However, the pronephros on the injected side (D) consists only of a single unbranched tubule and is
assigned only 1 point (d9). The PNI for this embryo is 4, again indicating inhibition of pronephric development. While the morphology of
the uninjected pronephros is normal (c9), note that it is somewhat swollen compared to normal, likely the result of hypertrophy in order
to compensate for loss of pronephric function on the injected side (Fox, 1956; Howland, 1921). The reduction in the amount of
b-galactosidase staining in injected embryos (B and D) compared to the embryo shown in Fig. 1 is consistent with the amount of
b-galactosidase mRNA injected: 0.25 ng co-injected with xWT1 in this figure compared to 1 ng when b-galactosidase was injected alone in
Fig. 1.
FIG. 3. Confocal microscopic analysis of the xWT1 overexpression phenotype. (A and B) High-magnification confocal optical sagittal
sections through the pronephroi of an embryo injected on one side with xWT1-KTS mRNA. The embryo is stained with 3G8 using a
rhodamine-conjugated secondary antibody, and the cell boundaries of tubular epithelial cells (indicated by arrows) are visible due to
autofluorescence of yolk platelets (see Wallingford et al., 1997). A single layer of pronephric epithelial cells surrounds the pronephric lumen
on both uninjected (A) and injected (B) sides of the embryos. However, the pronephric tubule epithelial complex on the injected side (B) is
significantly smaller than that on the uninjected side (A). (C and D) Confocal optical transverse sections of an embryo injected with
xWT1-KTS mRNA. The pronephros on the injected side (D) is significantly reduced compared to that on the uninjected side (C). The ventral
portion of the somite (S), the notochord (N), and the lateral plate (lp) are normal on both sides. The glomus (G) appears to be highly
disorganized on the injected side of the embryo (D), consistent with the hypothesis that signals from the tubules are required for the
formation of the glomus (Fales, 1935). (E and F) Confocal optical transverse sections of an embryo injected on one side with xWT1-KTS
mRNA and stained with propidium iodide to visualize nuclei. The pronephric tubule epithelium on the uninjected side (E, arrows) is
normal, while the tubule epithelium on the injected side (F, arrow) is significantly smaller and contains fewer cells.
FIG. 4. Injected xWT1 message persists throughout pronephric
development. RT–PCR using primers which specifically amplify
the injected message indicates that the injected xWT1 mRNA
remains stable until at least stage 30, by which time the pronephros
is well differentiated. EF1a was also amplified as a loading control.
Numbers at the top of the figure indicate developmental stages; 1,
injected embryos; 2, uninjected embryos; 2RT, no reverse transcriptase;
plasmid, PCR performed on 1 ng of xWT1 plasmid as a
positive control. The small amount of signal in the stage 10.5
uninjected sample is probably due to spillage during gel loading.
FIG. 5. xWT1 expression disrupts early events in pronephric specification. Expression of Xlim-1, an early molecular marker for the
pronephros, was detected at stage 20 by in situ hybridization (dark purple in all panels) and b-galactosidase was detected with X-Gal (light
blue/green in B and D). (A and B) Opposite sides of an embryo injected on one side with of b-galactosidase mRNA. Xlim-1 expression is
strong on both the uninjected (A) and injected (B) sides. (C and D) Opposite sides of an embryo injected on one side with xWT1-KTS
(1b-galactosidase mRNA). Xlim-1 expression is normal on the uninjected side (C), while expression is dramatically reduced on the injected
side (D). The result indicates that ectopic xWT1 mRNA expression inhibits pronephric development at a very early stage.
