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The pronephros functions in the amphibian larval stage. It differentiates in certain presumptive regions of the amphibian embryo. The study of molecules functioning during pronephrogenesis is important for understanding the mechanism of kidney formation. Herein, we report a gene expressed during differentiation of the pronephros and neural tissues that we isolated by differential hybridization using our pronephros in-vitro induction system. The gene, XCIRP, is 887bp in length, and encodes a putative protein composed of 163 amino acid residues. The deduced protein contains two CS-RBDs (consensus sequence RNA-binding domain) and a glycine-rich domain, and is 74% identical to homologs from other species (mouse, rat and human). The expression of XCIRP increased rapidly during gastrulation, and XCIRP localization was seen in the presumptive pronephros and neural tissues. These findings suggest that XCIRP may play important roles in pronephrogenesis and neurogenesis.
Fig. 1. Primary and secondary sequences of XCIRP. Nucleotide and deduced amino acid sequences of XCIRP cDNA (Accession No.: AB007597 in the DDBJ/EMBL/GenBank DNA Databases). The amino acid sequence is shown in single-letter code below the nucleotide sequence. The putative RNP motifs are underlined. The glycine-rich domain, well conserved among GRPs, is double-underlined. The stop codon is indicated by an asterisk.
Fig. 2. Comparison of amino acid sequences among CIRP homologs (Xenopus, rat, mouse, and human). The sequences that match perfectly are
enclosed in dotted boxes. The consensus sequence is shown at the top. XCIRP was 75% identical at the amino acid level to the mouse, rat, and
human homologs, and 74% identity at the amino acid level was demonstrated among the four homologs (mouse vs. rat: 100% identical, mouse or
rat vs. human: 95% identical at the amino acid level ).
Fig. 3. Northern blot analysis of XCIRP mRNA. RNA was prepared
from embryos at St. 6.5 ( lane 1), St. 9 (lane 2), St. 10 (lane 3), St. 12
( lane 4), St. 20 ( lane 5), St. 31 ( lane 6), St. 35 ( lane 7), St. 40 (lane
8). Autoradiography was performed overnight at −80°C. Expression
of ODC (ornithine decarboxylase) is indicated as a loading control.
Fig. 4. Whole-mount in-situ hybridization analysis of XCIRP transcripts in Xenopus embryos. (A) Lateral view of St. 9 embryo showing intensive
staining in the presumptive ectoderm. (B) Vegetal view of St. 11.5 embryo showing staining in all regions except the yolk plug. (C) Lateral view
of St. 20 embryo showing staining in the neural tube and neural crest. (D, E) Lateral views of St. 28 (D) and 32 (E) embryos showing extensive
staining in the neural region and pronephros rudiment. (F) Magnified view of the pronephric region in (E). Expression in the pronephros gradually
decreased from this stage onward. (G) Lateral views of St. 35 embryos. (H) Magnified view of the pronephric region in (G). Expression in
pronephric tubules has disappeared by this stage. ap, animal pole; vp, vegetal pole; yp, yolk plug; nt, neural tube; nc, neural crest; p, presumptive
pronephros; pt, pronephric tubules.
Fig. 5. Patterns of pronephrogenesis-related gene expression during Xenopus development. Patterns of pronephros marker gene, (Xlim-1, Xlcaax-
1, Na+, K+-ATPase a subunit, and XCIRP) expression, in the pronephric rudiment of normal embryos, are shown. The dotted area indicates the
period of expression of each marker gene. Symbols: −, no detection; ±, slight staining; +, staining; ++, extensive staining.
