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The periodic albino mutant of Xenopus laevis has been used to study the development of pigment cells because both the retinal pigment epithelium (RPE) and melanophores are affected. In this mutant, "white pigment cells" containing both melanophore-specific and iridophore-specific pigment organelles appear. The present experiments were designed to investigate the structural organization and expression of microphthalmia-associated transcription factor (Mitf) in the mutant since Mitf is known to regulate the development of melanocytes and RPE. The exon structures of X. laevis mitf genes (mitf.L and mitf.S) were defined using newly obtained Mitf transcripts and X. laevis genomic data. Compared to mouse mitf, exons 3 and 6a were absent in X. laevis mitf. The four exons between exons 4 and 6b in X. laevis mitf were named 5α, 5β, 5γ, and 5δ. Exons 5α and 5δ were specific to X. laevis mitf, whereas the continuous exons 5β/γ were identical to exon 5 of mouse mitf. A wide variety of A-Mitf and M-Mitf transcript variants lacking one or more exons were found in X. laevis; however, different types of Mitf transcripts were expressed in the mutant. In addition, white pigment cells and melanophores expressed both the mitf and dopachrome tautomerase (dct) genes.
Figure 1. Comparison of mitf exons between X. laevis and mouse. Exons used to produce AâMitf and MâMitf are indicated as boxes and numbered according to Hallsson et al. (). The exons present between exon 4 and exon 6b in X. laevis mitf were named 5α, 5β, 5γ, and 5δ. The continuous exons 5β and 5γ were thought to be identical to mouse mitf âexon 5â (see Figure for details). Both mitf.L and mitf.S differ from mouse mitf by the absence of exons 3 and 6a, and the presence of exons 5α and 5δ. Intron lengths (kb) in mouse mitf (Bharti, Liu, Csermely, Bertuzzi, & Arnheiter, ), mitf.L (NC_030730.1), and mitf.S (NC_030731.1) are shown.
Figure 2. Amino acid sequences of Mitf in X. laevis and mouse. The activation domain (AD), basic region, helixâloopâhelix domain, and leucine (L) zipper domain are shown. Identical residues and conserved residues are shaded black and gray, respectively. Arrows indicate the boundaries of exons 2a/b, 5β/γ, and 6a/b. Exons 3 and 6a are specific to mouse mitf, whereas exons 5α and 5δ are specific to X. laevis mitf. The continuous exons 5β/γ of X. laevis mitf are identical to exon 5 of mouse mitf. When Mitf transcript variants are produced by skipping exon 5δ, the amino acid changes from Lys (K) to Glu (E) in the beginning of exon 6b (asterisks) in both Mitf.L and Mitf.S (see Figure for details). Accession numbers: mouse AâMitf, NM_001113198.1; mouse MâMitf, NM_008601.3; X. laevis Mitf, LC315567âLC315583 (DDBJ).
Figure 3. Comparison of âexon 5â in mouse mitf and exons 5β/γ in X. laevis mitf.L and mitf.S. The amino acid sequences of exons 5β/γ in X. laevis mitf.L and mitf.S are identical to those of âexon 5â in mouse mitf (see Figure ). AGG (dark gray) and GCC (black) in mouse mitf âexon 5â correspond to AAG (green) in exon 5β and GTA (blue) in exon 5γ, respectively, in X. laevis mitf.L and mitf.S. If AGG (dark gray) and GCC (black) change into AAG (green) and GTA (blue), splice acceptor (green) and donor (blue) sites would be created, respectively. Arrows indicate the boundary of exon 5β and exon 5γ in X. laevis mitf.L and mitf.S.
Figure 4. Alteration of a single amino acid associated with exon skipping in transcript variants of Mitf.L (a) and Mitf.S (b). When exon 6b is linked to exon 5δ, the amino acid in the beginning of exon 6b is a Lys (K) in both Mitf.L and Mitf.S. However, in the transcripts skipping exon 5δ (Mitf.L Î5δ and Mitf.S Î5δ), the amino acid in the beginning of exon 6b is a Glu (E). These Mitf transcript variants skipping exon 5δ are present both in the wild type and the mutant (see Figure ).
Figure 5. Transcript variants of AâMitf (a) and MâMitf (b) in the wild type and the mutant. Exons are shown as boxes and numbered as in Figure . New transcript variants, AâMitf.L Î1b(CAG) and AâMitf.S Î1b(CAG), were present in AâMitf.L and AâMitf.S of the mutant. These variants were produced by alternative splicing at acceptor site (CAG), deleting the first 3âbp (CAG) of exon 1b (see Figure for details). The numbers on the right represent the number of clones with the transcript variant per the total number of clones sequenced. The data were collected and presented separately for the wild type and the mutant. Single asterisks indicate Mitf transcript variants observed only in the wild type. Double asterisks indicate Mitf transcript variants detected only in the mutant. Accession numbers: AâMitf transcript variants, LC315567âLC315575 (DDBJ); MâMitf transcript variants, LC315576âLC315583 (DDBJ).
Figure 6. Newly identified transcript variants, AâMitf.L Î1b(CAG) and AâMitf.S Î1b(CAG), produced by alternative splicing at the acceptor site (CAG) in exon 1b in the mutant. The DNA chromatogram, amino acid sequences, and splice site of exon 1b in AâMitf.L (a) and AâMitf.S (b) are shown. Subgenome genes, mitf.L and mitf.S, have the identical sequence around the boundary of intron and exon 1b, where tandem splice sites (CAGCAG acceptors) are present (c). There is no difference in the sequence around the acceptor site of exon 1b of the wild type and the mutant. Alternative splicing at the tandem acceptor (CAG) in exon 1b leads to the deletion of one amino acid, Ser (S), in both AâMitf.L and AâMitf.S.
Figure 7. Expression of mitf mRNA in white pigment cells and melanophores. RNA probes were generated from the sequence encoding either exons 1a–4 or exons 7–9 of Mitf.S (a). Photographs of the wildâ€type tail (b–e) and the mutant tail (f–i) at stage 46 are shown. Tadpoles were bleached to remove melanin before hybridization. In the negative control using a sense probe of either mitf 1a–4 or mitf 7–9, no staining was observed in the tails of either the wild type (b, c) or the mutant (f, g). WISH using an antisense probe of mitf 1a–4 (d, h) indicated that staining was observed in melanophores in the wildâ€type tail (d, arrowheads) and white pigment cells in the mutant tail (h, arrows). Using an antisense probe of mitf 7–9, staining was also detected in melanophores in the wildâ€type tail (e, arrowheads) and white pigment cells in the mutant tail (i, arrows). nc, notochord. sc, spinal cord. Scale bars: 100 μm.
Figure 8. Expression of dct mRNA in white pigment cells and melanophores. Photographs of the wildâ€type tail (a, b) and the mutant tail (c, d) at stage 46 are shown. Tadpoles were bleached to remove melanin before hybridization. In the negative control using a sense probe of dct, no staining was observed in the tails of either the wild type (a) or the mutant (c). Using an antisense probe of dct, staining was detected in melanophores in the wildâ€type tail (b, arrowheads) and in white pigment cells in the mutant tail (d, arrows). nc, notochord. sc, spinal cord. Scale bars: 100 μm.
