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???displayArticle.abstract??? Glial cell line-derived neurotrophic factor (GDNF) has multiple roles in kidney morphogenesis, spermatogenesis, and neurogenesis during development. In this study, we report the cloning and expression pattern of Xenopus laevis GDNF. The X. laevis GDNF cDNA sequence has a complete open reading frame of 684 bases, predicting 227 amino acid residues at the protein level. Comparison of the X. laevis GDNF amino acid sequence with those of chick, human, mouse, rat and zebrafish indicates that X. laevis GDNF has 60%-52% and 75%-62% identity over the whole amino acid sequence and for the putative mature forms, respectively. All known functional motifs of GDNF were conserved in the X. laevis sequence. Temporal expression analysis by RT-PCR indicated that GDNF transcripts were first detectable at stage 12 at a low level, and gradually increased up to stage 22. From stage 24, the expression sharply increased and continued at a similar level as development progressed. Spatial expression analysis by whole-mount in situ hybridization showed that the GDNF mRNA was predominantly detected in somites, pronephros, pharyngeal arches, epibranchial placodes, digestive tract and some of the lateral line structure. These results suggest that this X. laevis gene is the orthologue for GDNF.
Fig. 3. Spatial expression pattern of X. laevis GDNF in developing embryos. Whole-mount in situ hybridization analysis showed the localization of X. laevis GDNF transcripts. Lateral view at stage 22 (A), stage 24 (B), stage 26 (C), stage 31 (E), and stage 34 (H and I). Dorsal is up, and anterior is left. (D) The longitudinal section at stage 26. Anterior is left. (F and G) Transverse sections at stage 31 in the eye (F) and the pronephros (G) levels. Dorsal is up. (J and I) Digestive tracts dissected from stage 45 embryos hybridized with anti-sense (J) and sense (K) probe. Arrows and arrowheads indicate pronephros and pharyngeal arches, respectively. The dotted line in (C) illustrates the plane of section in (D). The dotted lines through the eye and the pronephros in (E) correspond to planes of section in (F) and (G), respectively. Abbreviations: E, eye; IX, glossopharyngeal epibranchial placode; NT, neural tube; pAD, anterodorsal lateral line placode; pM, middle lateral line placode; pP, posteriorlateral line placode; VII/pAV, a composite structure of the facial epibranchial placode and the anteroventral lateral line placode. Scale bar is 500Â micrometers.
Fig. 2. Temporal expression pattern of X. laevis GDNF during development. RT-PCR analysis showed the expression pattern of X. laevis GDNF transcripts in unfertilised egg (U.F.) and embryo stages (St.). The amount of input cDNA was confirmed by PCR with ornithine decarboxylase primers (ODC RT+). Contamination of genomic DNA was examined by experiments without RT reactions (ODC RTô°Š). Stage 22 cDNA was used for the linearity control. The black triangle illustrates doubling input of the cDNA to ensure linearity of the amplification.
gdnf (glial cell derived neurotrophic factor) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 22 lateral view, anteriorleft, dorsal up.
gdnf (glial cell derived neurotrophic factor) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 24, lateral view, anteriorleft, dorsal up.
gdnf (glial cell derived neurotrophic factor) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 26, (right) lateral view, anteriorleft, dorsal up, and (left) horizontal section as indicated (D).
