Shimizu-Nishikawa K et al. (1999), Expression of helix-loop-helix type negative re...

XB-ART-11603

Dev Growth Differ 1999 Dec 01;416:731-43. doi: 10.1046/j.1440-169x.1999.00477.x.

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Expression of helix-loop-helix type negative regulators of differentiation during limb regeneration in urodeles and anurans.

Shimizu-Nishikawa K , Tazawa I , Uchiyama K , Yoshizato K .

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The urodele is capable of regenerating its limb by forming a blastema even in the adult. By contrast, the anuran, which is phylogenetically close to the urodele, loses this ability during metamorphosis and forms blastema-like tissues that develop only into a spike-like structure in the adult. In order to compare the molecular mechanism of the formation and maintenance of the blastema between the urodele and anuran, the genes encoding helix-loop-helix (HLH) type negative regulators of differentiation were characterized for both the Japanese newt, Cynops pyrrhogaster, and African clawed frog, Xenopus laevis. Cynops homologs of Id2, Id3, and HES1 and Xenopus Id2 were identified. To learn the roles of these genes in regeneration, their expression was examined. The expression of Id2 and Id3 was low in unamputated limbs, but was up-regulated in blastemas of both adult newt and Xenopus. Interestingly, transcripts of the two Id genes showed specific localizations in the blastema and the expression patterns were very similar in both species through the early to medium bud stage. Id2 was expressed predominantly in the blastemal epidermis, and Id3 was expressed equally in the blastemal epidermis and mesenchyme including cells in precartilage condensations. HES1 expression was up-regulated in the newt blastemal epidermis. It was thought that the up-regulation of these genes in the epidermis was related to the proliferation of the cells and that increased expression of these genes in the mesenchyme was related to the undifferentiated state of the blastemal cells. These results and considerations strongly suggested that the state of differentiation is similar in the early to medium bud blastema of both urodeles and anurans. The expression of Id3 remained high through to the digits stage in newts. In contrast, its expression in Xenopus decreased in spike-like regenerates, which correspond to palette-digits stage of newt regenerates. From these results, it was suggested that the blastema redifferentiates earlier in the frog than in the newt, and therefore the timing of redifferentiation of the cartilage is crucial for complete regeneration.

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Species referenced: Xenopus laevis
Genes referenced: hes1 id2 id3 id4 tbx2

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	Fig. 1. Comparison of the predicted amino acid sequence. (A) Id2, (B) Id3, and (C) HES1. Amino acid residues identical to the newt were replaced by asterisks and gaps (dashes) were introduced to improve alignment. In the newt HES1 sequence, the proline residue in the basic region is double underlined and the boxed sequence indicates the conserved amino acid sequence in the C-terminal region. Sequence references; Sun et al. (1991) for mouse Id2; Christy et al. 1991 for mouse Id3; Wilson and Mohun 1995 for Xenopus Idx; and Takebayashi et al. (1994) for mouse HES1. (D) Phylogenetic tree of Id genes. Sequence references: Benezra et al. 1990 for mouse Id1; Sun et al. 1991 for mouse Id2; Christy et al. 1991 for mouse Id3; Riechmann et al. (1994) for mouse Id4; Hara et al. (1994) for human Id1; Biggs et al. (1992) for human Id2; Ellmeier et al. (1992) for human Id3; Kiesling T. L., Swiss-prot: P47928 for human Id4; Wilson and Mohun 1995 for Xenopus Idx; and Ellis et al. (1990) for Drosophila emc.
	Fig. 2. Expression of Id and HES1 in limbs of the newt and Xenopus. Ten micrograms of total RNA was loaded to each lane. The ethidium bromide (EB) staining pattern was shown in the bottom panel for each set of data to confirm that the same amount of RNA was loaded to each lane. Probes used for hybridization are indicated in the left side of each panel. (A) Expression of Id and HES1 in the newt. Stages of regeneration are indicated at the top of the panel; (Normal) unamputated normal limbs, (Early bud) blastemas of early bud stage, (Medium bud) medium bud stage, (Late bud) late bud stage, (Palette) regenerates of palette stage, (Digits) digits stage. (B) Id expression in Xenopus. Developmental stage and the name of the limb parts are indicated at the top of the panel. Left panel; Normal, unamputated normal limb buds at stage 56; Regenerate, regenerates that were amputated at stage 55 and collected 1 week after the amputation (when normal siblings proceeded to stage 56); Norm. prox., proximal part of unamputated normal limbs of adult; Norm. dis., distal part of unamputated normal limbs of adult; Blastema, blastemas of adult frogs 16 days after amputation (newt regenerates developed to early to medium bud stage during this period). Right panel; Normal, unamputated normal limbs of adult frog; blastema, blastemas of adult frog 16 days after amputation (same sample as in the left panel); Spike, spike-like regenerates of adult frogs 52 days after amputation (newt regenerates developed to palette to digits stage during this period). (C) Id expression in the developing limbs of Xenopus. Developmental stages are indicated at the top of the panel. (D,E) Localized expression of Id and HES1 in the blastema. Total RNA was isolated from the epidermis and the muscle of normal (unamputated) limbs, and the epidermis and the mesenchyme of blastemas of the earlymedium bud stage. The tissues for the RNA source are indicated at the top of the panel. (D) Newt. (E) Xenopus. In the northern analysis of adult Xenopus tissue, two bands were detected in only one case (B, right panel) out of three cases (B, right and left panels, and E). The sample shown in Fig. 2(B) right panel was collected from 14 individual frogs. It is likely that the extra bands are the result of individual variants.
	Fig. 3. Identification of Id gene expressing cells in the blastema. The expression of the Id family was examined by in situ hybridization. (A,B) Id2 expression in the late bud stage of the newt blastema. (C,D) Id3 expression in the late bud stage of the newt blastema. (E,F) Id2 expression in early-medium bud stage of the Xenopus blastema. (G,H) Id3 expression in early-medium bud stage of the Xenopus blastema. Anti-sense probes were used for the detection of transcripts in (A,C,E,G) and sense probes in (B,D,F,H). Large closed arrowheads in (A,E) indicate the Id2- expressing epidermal cells. Open arrowheads in (A) indicate the Id- 2-expressing mesenchymal cells near the wound epidermis. Small closed arrowheads in (C,G) indicate the Id3-expressing cells in precartilage condensations. Bar, 200 μm.