Cadinouche MZ et al. (1999), Molecular cloning of the Notophthalmus viridesc...

XB-ART-13325

Dev Dyn 1999 Mar 01;2143:259-68. doi: 10.1002/(SICI)1097-0177(199903)214:3<259::AID-AJA9>3.0.CO;2-G.

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Molecular cloning of the Notophthalmus viridescens radical fringe cDNA and characterization of its expression during forelimb development and adult forelimb regeneration.

Cadinouche MZ , Liversage RA , Muller W , Tsilfidis C .

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Larval and adult newts provide important experimental models to study limb development and regeneration. These animals have exceptional ability to regenerate their appendages, as well as other vital structures. Our research examines the role of the fringe gene (fng) in the developing and regenerating adult newt forelimb. Fringe codes for a secretory protein. It was first discovered in Drosophila, and later homologues were isolated in Xenopus laevis, chick and mouse. This gene has been highly conserved throughout evolution, indicating its crucial role in vertebrate and invertebrate development. We have isolated, cloned, and sequenced the full length of the Notophthalmus viridescens radical fringe cDNA (nrFng) by screening a newt forelimb blastema cDNA library with a 500-bp fragment of the Xenopus lunatic fringe cDNA. The newt fringe cDNA codes for a 396 amino acid protein with a predicted N-terminal signal sequence. Newt fringe shows high homology with radical fringe homologues of many species. Whole mount mRNA in situ hybridization on several stages of newt limb development reveals that nrFng is first expressed in the limb field, with intense expression as the limb bud develops. However, gene expression diminishes with more advanced digit development. A significant role in adult forelimb regeneration is also evident, as we isolated the cDNA from a regeneration-specific library and found it highly expressed during the regenerative phases of active cell division and then down regulated at sites undergoing differentiation and morphogenesis.

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Species referenced: Xenopus laevis
Genes referenced: dio3 rfng

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Fig. 3. nrFng expression in developing forelimbs of Notophthalmus viridescens embryos. Expression is first seen in the presumptive forelimb field before the limb bud emerges (a). As the limb bud develops (b,c) and elongates to form a cone (d), expression is very intense in the mesen- chyme regions of the developing limb. A homogeneous signal spanning both dorsal and ventral halves of the developing forelimb is seen. Strong nrFng expression persists as the cone flattens into a palette and digitation begins to appear (f,g). This expression is clearly found in the mesodermal tissues and not the ectoderm, as shown in parts b and e. At the three digit stage (h), the strength of nrFng expression diminishes. By the early four-digit stage (i), nrFng expression is no longer detectable in the forelimb. A comparison between sense (j, embryo on left) and antisense (j, embryo on right) signal shows the specificity of the probe. EG, external gill; LF, limb field; E, ectoderm; M, mesoderm; LB, limb bud; C, cone; P, pigment; D1, D2, D3, D4, digits 1. Scale bar = approx. 200 μm.

Fig. 5. nrFng expression in regenerating adult newt forelimbs. The black lines show the approximate level of amputation. Expression is not seen in the early regenerate (a) prior to the formation of the blastema. As the regenerate develops, nrFng expression is weakly seen in the blastema (b), as confirmed by RT-PCR results, and then becomes more intense in the early digit (c) and mid-digit (d) stages. Expression begins to decrease in the advanced digit stage (e). Examination of Figures c, d, and e clearly shows that expression is absent in the overlying epithelium, and is concentrated in the mesenchymal regions between the differentiating cartilagenous digits. The sense control is seen in (f). C', cartilage; IM, interdigital mesenchyme. Magnification x100.