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Vertebrates use two or three forms of kidney successively during development and the nephric duct is essential for this succession of kidney induction. While transcripts of many Wnt ligands and Wnt receptor Frizzled genes have been localized in developing kidney, the relationship between Wnt signaling and nephric duct development remains unknown. This study investigated the role of Xenopus frizzled-8 (Xfz8) in pronephros development. Translational inhibition of Xfz8 caused a significant reduction in the staining of a duct-specific antibody, but did not affect the expression of early pronephric maker genes in the duct region. Defects in pronephric tubule branching were also observed following inhibition of Xfz8. Histological analysis revealed that the Xfz8-inhibited cells failed to form a normal epithelium structure. These results suggest that Xfz8 is involved in the process of normal epithelium formation in the developing pronephric duct and tubules after specification.
Fig. 1. Spatial localization of Xfz8 transcripts during pronephric
development was visualized by whole-mount in situ hybridization
analysis. The expression of Xfz8 (F–J) was compared with that of
Xlim-1 (A–E). (A,F) At stage 18, expression of Xfz8 was only observed
in the prechordal region and not in pronephric precursors. Dorsal
midlines, anterior (A), and posterior (P) are indicated. (B,G) At stage
22, slight expression of Xfz8 was observed in the pronephric anlage,
while Xlim-1 was strongly expressed. The arrowhead indicates Xfz8
staining in the pronephric anlage. (C,H) At stage 26, the expression of
Xfz8 increased. (D,I) At stage 28, localization of Xfz8 transcripts was
restricted to the duct region of the pronephros, while Xlim-1 was
observed in both nephrostomes and the duct region. (E,J) At stage 33,
Xfz8 continued to be expressed in the pronephric duct region.
Fig. 2. Activity of Xfz8 antisense morpholino oligonucleotide (Xfz8-
Mo). (B) Xfz8-eGFP mRNA (500 pg), (C) Xfz8-eGFP mRNA
(500 pg) + Xfz8-Mo (20ng), (D) d7n-Xfz8-eGFP mRNA (500 pg), and
(E) d7n-Xfz8-eGFP mRNA (500 pg) + Xfz8-Mo (20ng) were injected
in the animal pole of Xenopus embryos. The embryos were raised until
stage 10 and the eGFP was visualized. Xfz8-Mo inhibited Xfz8-eGFP
but not d7n-Xfz8-eGFP. (F) Embryos injected with Xfz8-Mo (20 ng) in
both lateral sides developed edema, a characteristic of renal defect.
Fig. 3. Whole-mount immunohistochemistry analysis. (A) Control
Mo (20 ng), (B,D) Xfz8-Mo (20 ng), and (C) Xfz8-Mo (20 ng) + d7n-
Xfz8/pCS2 (5 pg) were injected into the left side of the embryos. The
embryos were cultured until stage 40 and subjected to 4A6 and 3G8
antibodies (AâC) or 4A6, 3G8, and 12/101 antibodies (D). Pronephric
duct staining with 4A6 was significantly reduced by Xfz8-Mo (arrows
in B), but rescued by co-injection with d7n-Xfz8/pCS2 (C,E). The
staining pattern of the pronephric tubule with 3G8 was also changed.
Normal tubules have three branches as indicated by the arrowheads in
(A), but the branches were shorter or reduced in number in the Xfz8-
Mo-injected side (arrowheads in B). The tubule defects were not
rescued by co-injection with d7n-Xfz8/ pCS2 (C). 12/101 staining was
not altered by Xfz8-Mo (D). (E) Frequency of 4A6 or 3G8 staining
patterns observed in the injected side of embryos.
Fig. 4. Whole-mount in situ hybridization analysis. Xfz8-Mo (20 ng) with b-galactosidase mRNA (300 pg) were injected into one lateral side of
albino embryos. These embryos were raised until stage 33 and b-galactosidase activity was visualized with Red-gal substrate as a tracer. The embryos
were then subjected to whole-mount in situ hybridization with probes, Xlim-1 (A), Xpax-2 (B), Claudin (C) or Na+, K+-ATPase (D). The expression
of Xlim-1, Xpax-2, Claudin or Na+, K+-ATPase was not reduced in the Xfz8-Mo-injected side but several embryos showed massive staining in the
tubule region indicated by an orange arrowhead in (B). Black arrowheads indicate three nephrostomes and arrows indicate pronephric duct.
Fig. 5. Transverse sections of the pronephros. Embryos after 4A6 and
3G8 staining were sectioned. The apical surface of pronephric tubule
structures was stained with 3G8 (red arrow). The entire cell surface of
the duct structure was stained positive with 4A6 (red arrowhead).
Unstained duct structures were observed in the Xfz8-Mo-injected side
(B, arrowheads). In the Xfz8-Mo + d7n-Xfz8/pCS2-injected side, 4A6
staining was partially rescued (C). (DâF) Hematoxylin and eosin
staining was performed on the same section of (A, B, and C),
respectively. (A,D) Normal duct and tubule cells were cubic in shape
and were arranged regularly. (B,E) In the Xfz8-Mo-injected side, duct
and tubule cells were circular in shape and arranged irregularly. (C,F)
Co-injection of d7n-Xfz8/pCS2 partially rescued the defects caused by
Xfz8-Mo. (GâI) Duct structures in the uninjected, Xfz8-Mo-injected or
Xfz8-Mo + d7n-Xfz8/pCS2-injected side of a section at the caudal
position, respectively. (JâL) Hematoxylin and eosin staining was
performed on the same caudal section (G, H, or I), respectively. (G,J)
Normal duct structure. (H,K) Duct cells in the Xfz8-Mo-injected side
were circular in shape and arranged irregularly. (I,L) Co-injection of
d7n-Xfz8/pCS2 partially rescued the defects caused by Xfz8-Mo.
