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We describe the isolation and characterization of the RNA-binding protein XC3H-3b that is expressed during pronephros development. XC3H-3b is a member of the TTP/TIS family of CCCH tandem zinc-finger proteins, which are physiological stimulators of instability for the mRNA encoding tumor necrosis factor-alpha in certain cell types. XC3H-3b is localized primarily to the mesodermal tissues around the pronephros. Overexpression of XC3H-3b markedly and specifically inhibits kidney development. Morpholino-mediated knockdown of XC3H-3b also results in defects in nephrogenesis. In both cases, the expression of numerous pronephric marker genes, such as Xlim-1, Xpax-2, Xpax-8, Xwnt-4, and XWT1, is decreased and morphological development of the pronephric tubules is abrogated. We conclude that XC3H-3b plays an important role in the regulation of pronephros differentiation. This is the first report of a gene localized around the pronephros that regulates pronephros development.
Fig. 1. Alignment of the deduced amino acid sequences of Xenopus XC3H-3b, XC3H-3, and mouse and human TIS11b. The underlined sequences
represent the two CCCH zinc-finger domains in XC3H-3b. XC3H-3b shared 88% sequence identity in TIS11b/c N-terminal domain (36â110 aa) with
XC3H-3, the paralogue of XC3H-3b. XC3H-3b also shared 52% identity with mouse TIS11b and 53% identity with human TIS11b sequence.
Fig. 2. Expression of XC3H-3b during Xenopus development. (A) Temporal expression of XC3H-3b was analyzed by RT-PCR from the unfertilized
egg to larval stage (stage 40). RT, control reverse transcription without transcriptase. (B–F) Expression of XC3H-3b in mesodermal tissues around
the pronephric region of the embryo. (B) Beginning in the late neurula stage, transcripts of XC3H-3b were detected in the anterior crest and mesodermal
tissues around the pronephros. (C) Anterior view at stage 21. (D) Expression patterns of XC3H-3b in a tail-bud stage embryo. This XC3H-
3b expression pattern was maintained until the tadpole stage. (E,F) Lateral view of tadpole embryos. Arrows indicate the position of pronephros. ba,
branchial arches; ha, hyoid arch; ma, mandibular arch; mh, mid-hindbrain boundary; and ov, otic vesicle. (G) Histology section of Xenopus embryo
showed in Fig. 3D. Arrows indicate the expression of XC3H-3b localized in the mesodermal tissues around pronephros.
Fig. 3. Microinjection was performed into one side of C2 and C3
blastomeres of 32-cell stage embryos. (AâD) Embryos were fixed at
stage 40 and then development of the pronephros was analyzed by
immunohistochemistry staining with monoclonal antibodies 3G8 for
tubules and 4A6 for ducts. Arrows indicate the position of pronephros.
(A) Expression of XC3H-3b inhibited the development of
the pronephros. Five hundred picograms of XC3H-3b mRNA and
250 pg b-galactosidase mRNA were injected into one side of C2 and
C3 blastomeres of 32-cell stage embryos. Pronephric tubule formation
was obviously inhibited in XC3H-3b-injected embryos. (B) Ten
nanograms of MO (XC3MO) and 250 pg b-galactosidase mRNA
were injected into C2 and C3 blastomeres of 32-cell stage embryos.
Pronephros was inhibited in XC3MO-injected embryos. (C) The 5-
mis MO-injected embryos. Pronephric tubules developed normally in
these embryos and duct can be observed. (D) The b-galactosidaseinjected
embryos. Pronephric tubules developed normally in these
embryos and duct can be observed. (E,F) The histological analysis
of XC3H3-b- and XC3MO-injected embryos. (E) Pronephric tubules
in XC3H3-b-injected embryos show developmental defects. (F)
Pronephric tubules and ducts in XC3MO-injected embryos show
severe developmental defects. Arrows indicate the hypoplasia of
pronephric tubules. (G) Sequence-specific inhibition of XC3H-3b
translation by XC3MO. We assayed whether the XC3MO inhibited
the translation of XC3H-3b in a sequence-specific manner. XC3MO
was injected with 100 pg XC3H-3b-6myc into Xenopus embryos.
Significant reduction of XC3H-3b-6myc protein synthesis was observed
in XC3H-3b-6myc/XC3MO co-injected embryos. The effect of
5-mis MO was also tested and injection of 5-mis MO did not inhibit
protein synthesis. The dosages of MO were 20 ng/embryo. Scale bar,
50 lm.
Fig. 4. Suppression of pronephros-specific gene expression in XC3H-3b- and XC3MO-injected embryos. Embryos were co-injected with 500 pg
mRNA of XC3H-3b or 10 ng XC3MO (XC3H-3b: A, E, I, M, Q, U); XC3MO: B, F, J, N, R, V) and 250 pg b-galactosidase, fixed at stage 30/31, and
the activity of b-galactosidase was measured. Control embryos were injected with 500 pg b-galactosidase (D,H,L,P,T,X). In addition, embryos were
injected with 10 ng of 5-mis MO to evaluate the MO specificity (C,G,K,O,S,W). Expression of pronephros-specific genes was detected by wholemount
in situ hybridization. (A) Expression of Xlim-1 was unaffected by injection of XC3H-3b, but was reduced by injection of XC3MO. (B).
Expression of Xlim-1 was observed in tubular and duct regions. Reduced expression of Xlim-1 was observed in tubular but not in duct regions.
Expression of Xlim-1 in the duct was not affected by injection of XC3MO. (E) No obvious down-regulation of Pax-2 expression was observed in
XC3H-3b-injected embryos. (F) Down-regulation of Pax-2 expression was observed in XC3MO-injected embryos. (IâJ,MâN) Expressions of Pax-8
and Wnt-4 were inhibited by injection of XC3H-3b and XC3MO. Expressions of these two genes were markedly suppressed in both the tubular and
ductal regions. (Q,R) Expression of the glomus marker gene, WT1, was analyzed. Down-regulation of WT1 expression was observed in XC3MO-
(R), but not in XC3H-3b-injected embryos (Q). (UâX) Expression of mesodermal marker, XmyoD in XC3H-3b-, XC3MO-, 5-mis MO-, and bgalactosidase-
injected embryos. Expression of XmyoD was not affected by injection of XC3H-3b (U) and XC3MO (V).
