Ishino T et al. (2003), Identification of genes induced in regenerating...

XB-ART-5826

Dev Dyn 2003 Feb 01;2262:317-25. doi: 10.1002/dvdy.10229.

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Identification of genes induced in regenerating Xenopus tadpole tails by using the differential display method.

Ishino T , Shirai M , Kunieda T , Sekimizu K , Natori S , Kubo T .

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To identify candidate gene(s) involved in the tail regeneration of Xenopus laevis tadpoles, we used the differential display method to isolate four genes (clones 1, 2, 13a, and 13b) whose expression is induced in regenerating tadpole tails. Among them, clones 13a and 13b were found to encode the Xenopus homologues of the alpha1 chain of type XVIII collagen and neuronal pentraxin I, respectively. Expression of clone 2 and neuronal pentraxin I genes increased dramatically in the blastema 3 days after amputation, whereas that for the clone 1 and type XVIII collagen genes was induced gradually after amputation. In situ hybridization revealed that the neuronal pentraxin I gene is expressed specifically in the regenerating tail epidermis but not in the normal tail epidermis or the most distal margin of the tail blastema, suggesting that it has a tissue-inductive role in tail regeneration. Expression of the four genes was induced in the limb and in the tail blastema, suggesting that they are involved in the regeneration of both organs. Finally, expression of clone 2 and neuronal pentraxin I genes was scarce during embryonic stages in comparison to the tail blastema, suggesting that their main functions are in organ regeneration. Our results demonstrate unique features of spatial and temporal gene expression patterns during Xenopus tadpole tail regeneration.

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Species referenced: Xenopus laevis
Genes referenced: col18a1 hoxa1 hoxa11 nptx1

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	Fig. 6. Whole-mount in situ hybridization of the Xenopus NP1 gene in the regenerating tadpole tails. A,B: Whole-mount in situ hybridization was performed using Xenopus tadpole regenerating tails 7 days after amputation and antisense (A) or sense (B) digoxigenin- labeled RNA probes. The boundary between the normal tail portions and the regenerating tail is indicated by white arrowheads, and the boundary between the regenerating tail and the most distal portions is indicated by black arrowheads. C: The tadpole regenerating tail specimen stained by whole-mount in situ hybridization was sectioned to observe the inner tissues. The boundary between the most distal tip without NP1 expression (right) and the NP1-expressing regenerating tail (left) is indicated by filled red arrowheads. The portion corresponding to the distal tip is outlined by open red arrowheads.
	Fig. 3. Northern blot hybridization of the type XVIII collagen and NP1 mRNAs. A total of 10 g of total RNA from regenerating tail (R) or from normal tail (N) was subjected to Northern blotting using cDNAs for Xenopus type XVIII collagen (left upper panel) and NP1 (right upper panel) as probes. Lower panels: The blots were hybridized to an EF1 clone to check whether each lane contained an equal amount of RNA. Positions of RNA makers (Boehringer Mannheim) in the base are indicated at the right of the upper panels. The arrowhead on the left of each panel indicates the position of signals.
	Fig. 4. Time-course of the expression of the four genes after tail amputation. A: Reverse transcription-polymerase chain reaction (RT-PCR) analysis of expression of each gene in the regenerating tadpole tails 0, 1, 3, 5, and 7 days after the amputation. PCR cycles used were as follows: 24 for clone 1 and Xenopus type XVIII collagen genes; 26 for the clone 2 and Xenopus NP1 genes; 16 for the Xenopus EF1 gene. B: RT-PCR analysis of expression of the HoxA1, HoxA11, Hox36, and EF1; gene in the intact (I) or regenerating tadpole tails (R). Regenerating tails were recovered 7 days after amputation.
	Fig. 5. Region-specific expression of the four genes in the regenerating tadpole tails. A: Schematic diagram representing the operation and tissues used to analyze the expression patterns of the four genes. a, the anterior portion of the amputated tail; b, the most proximal part of the regenerating tail; c, the neighboring part to the blastema; d, the blastema. B: Reverse transcription- polymerase chain reaction (RT-PCR) analysis of expression of each gene in the regenerating tadpole tails. a�d corresponds to the parts outlined in A. PCR cycles used were as follows: 24 for clone 1 and Xenopus type XVIII collagen genes; 26 for the clone 2 and Xenopus NP1 genes; 16 for the Xenopus EF1 gene.
	Fig. 7. The expression of the four genes in the regenerating tadpole hindlimbs. The expression of each gene in the normal (N) or regenerating tadpole hindlimbs (R) were analyzed by reverse transcription-polymerase chain reaction experiments.
	Fig. 8. Expression of the four genes during normal Xenopus development. Reverse transcription-polymerase chain reaction (RT-PCR) analysis was performed by using total RNAs extracted from various stages of embryos, tadpoles, and adults with specific primers for each 4 genes and the EF1 gene as a control. PCR cycles used were as follows: 27 for the clone 1, 2, and Xenopus type XVIII collagen gene; 24 for the Xenopus NP1 gene; 17 for the Xenopus EF1 gene.