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First published online 13 February 2008
doi: 10.1242/dev.014142

Development 135, 1169-1178 (2008)
Published by The Company of Biologists 2008
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Alternative promoter use in eye development: the complex role and regulation of the transcription factor MITF

Kapil Bharti, Wenfang Liu, Tamas Csermely*, Stefano Bertuzzi and Heinz Arnheiter{dagger}

Mammalian Development Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892, USA.


Figure 1
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  		Fig. 1. Generation and structure of MITF isoforms in mouse. (Top) Schematic of the mouse Mitf gene with its multiple promoters and regular and alternative splice choices. (Bottom) Schematic of the seven protein isoforms that differ in their N-terminal sequences owing to different promoter choice.

 

Figure 2
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  		Fig. 2. Expression of Mitf RNAs and proteins containing exon 1B1b. (A) In situ hybridization with an exon 1B1b-specific probe on frontal sections of the developing mouse eye at the indicated time points. Arrowheads at E9.5 indicate expression of exon 1B1b-containing Mitf RNAs throughout the optic vesicle, and the arrowhead at E10.5 indicates reduced expression in the distal optic vesicle. In a control section of an Mitfmi-rw/mi-rw embryo, which lacks exon 1B1b, there is no labeling (right-most panel). (B) Antibody characterization and Mitf expression in RPE/choroid and retina. A-, H-, D-, and M-Mitf cDNAs were expressed in NIH3T3 cells and extracts blotted with polyclonal anti-1B1b MITF antibodies (top panel) or monoclonal anti-MITF C-terminal-domain antibodies (anti-CTD, middle panel). The bottom left panel shows a combined immunoprecipitation (IP)/immunoblot (IB) of NIH-3T3 cells transfected with A-Mitf. The bottom right panel shows a similar IP/IB of extracts from RPE/choroidal and retinal fractions from 100 wild-type eye primordia (E12.5). Arrowheads point to MITF isoforms with distinct electrophoretic mobilities. (C) Immunohistochemistry of frontal cryosections of embryos of the indicated genotypes and ages using anti-1B1b MITF and anti-pan-MITF antibodies. Arrows in the panel labeled wt E15.5 point to M-MITF-expressing neural crest-derived melanocytes that are only seen with pan-MITF-specific antibodies. Note that `wt' refers to Tyrc (albino) embryos carrying a wild-type Mitf allele. RPE, retinal pigment epithelium; ret: retina; CMZ, ciliary margin zone.

 

Figure 3
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  		Fig. 3. Differential expression of Mitf isoforms during mouse eye development. (A) RT-PCR analysis on RNA isolated from wild-type whole optic vesicles (E9.5-10.5) or separate RPE+choroidal and retinal fractions (E11.5-P0). (B) RT-PCR (left, lanes 1-6) and real-time PCR (right) for RNA isolated from Mitfmi-bw/mi-bw eyes, which lack neural crest-derived melanocytes. (Lanes 1-3) E15.5 eyes were dissected as in A and 100 individual cells were microscopically selected as described in Materials and methods. Lane 1, mesenchymal cells; lane 2, RPE cells; lane 3, retinal cells. The asterisk in lane 2 indicates that the corresponding cDNA was diluted 1:10 for the reaction with pan-specific primers. (Lanes 4-6) Pooled fractions from dissected E15.5 eyes as indicated. Lane 4, mesenchymal fraction; lane 5, RPE/mesenchymal fraction; lane 6, retinal fraction. (Right) Real-time PCR. RNA was prepared from separately pooled RPE/mesenchymal and retinal fractions from E11.5 and E15.5 eyes. Results are expressed as mean of absolute amounts of the respective cDNAs and are calculated taking into account that RPE cells represent 7% of the cells in the RPE/mesenchymal fraction. Significance of the difference between RPE and retina (P-values, Student's t-test; pools of 20 RPE/mesenchymal and retinal fractions each for E11.5, and of 12 RPE/mesenchymal and retinal fractions each for E15.5; four separate assays in triplicate per pool) for E11.5 A-Mitf, >0.1; J-Mitf, <0.1; H-Mitf, <0.001; D-Mitf, <0.00001; and for E15.5 A-Mitf, <0.01; J-Mitf, <0.01; H-Mitf, <0.0001; D-Mitf, <0.001.

 

Figure 4
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  		Fig. 4. Mitfmi-rw/mi-rw mutant mice carry a genomic deletion in Mitf encompassing the exons 1H, 1D and 1B. (A) Phenotype of an Mitfmi-rw/mi-rw mutant mouse. Generally, coat pigment patches and eye size are not correlated, as eye size is determined by the development of the RPE and coat patches by neural crest-derived melanocytes. To the right is shown the DNA sequence flanking the genomic deletion, with the numbers of the first and last base referring to the positions on chromosome 6 according to assembly NCBIM36. The schematic beneath shows the extent of the deletion and highlights the novel splice junctions that are generated between the upstream exons 1A, 1J, 1C, 1MC and 1E and the downstream exon 2A. For details, see text. (B) RT-PCR using RNA from E12.5 wild-type and Mitfmi-rw (rw) mutant embryos and the corresponding P0 newborns. Note increased band intensity in rw with exons 1A, 1J and 1E, but decreased band intensity at E12.5 with pan-specific primers (exon 9). (C) Real-time PCR from RNA of whole eyes harvested at the indicated ages, using primers as in B. The results are expressed as RNA levels in rw mutants relative to those in corresponding wild-type embryos (groups of 14 eyes each, three measurements each in triplicate). The increases in rw over wild type in 1A and 1J, as well as the decrease in exon 9 at E12.5, are statistically significant (P<0.01, Student's t-test). P-values for the increase in 1E are <0.02 at E12.5 and =0.13 at P0. No significant difference was found for exon 9 at P0 (P=0.27). (D) RT-PCR using primers spanning four exons. Note the differently sized products in wild type and rw for isoform 1A. For isoform 1J, the expected larger product in wild type is not seen because its relative level is low (see Fig. 2), but the smaller sized product in rw is visible. In 1E-4, the white arrowhead points to the correct E-Mitf band.

 

Figure 5
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  		Fig. 5. The Mitfmi-rw allele allows for the expression of Mitf and its target gene tyrosinase in the anterior RPE as well as for residual pigmentation in the iris but leads to an abnormal RPE dorsally. Sections of wild-type (wt) and Mitfmi-rw/mi-rw mutant mouse eyes of the indicated ages were stained with a pan-Mitf in situ probe (A,B,E,F), a pan-MITF antiserum (C,D,G,H), antibodies against tyrosinase (I-L), or against PAX6 (green) and TUJ1 (red) (Q-T). Note that the mutant eye expresses Mitf RNA (B,F) and low levels of MITF protein, along with tyrosinase, particularly at later stages (compare G with H, arrows; K with L). Also note that in the mutant at E17.5, the distal ciliary margin, although positive for Mitf RNA (F), is relatively free of MITF and tyrosinase (arrowhead in H and L, compare with arrowhead in G and K). (M,N) Strong pigmentation at P0 in wild type (M) and weak pigmentation in rw (N). (O,P) Pigmentation in adult wild-type iris (O) and rw iris (P). (Q,R) Dorsal thickening of the E12.5 RPE in rw (R) compared with wild type (Q). (S,T) RPE abnormalities in rw at E17.5. Arrowheads in S mark the location of the wild-type RPE, which now is free of PAX6 staining. By contrast, mutant RPE retains PAX6 staining and in this eye showed epihelial folds rather than homogeneous thickening (arrowhead in T).

 

Figure 6
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  		Fig. 6. Lack of downregulation of Mitf in Chx10orJ/orJ retinas predominantly affects H- and D-Mitf. (A) Genetic pathway showing the downregulation of Mitf in the future retina by FGFs emanating from the surface ectoderm (light blue) and Chx10 operating in the distal optic neurepithelium. In Chx10 mutant mice, Mitf is not downregulated in the future retina and the retina hypoproliferates. (B-E) Cryostat sections from E13.5 wild-type (wt) (B,D) and Chx10orJ/orJ (C,E) eyes labeled for phosphohistone H3 (B,C) or by in situ hybridization using an Mitf exon 1B1b probe (D,E). Note upregulation of exon-1B1b-containing RNA in mutant (E) compared with wild-type (D) retina. Also note that the section in E comes from an embryo with a pigmented RPE (brown stain), whereas the one in D comes from an albino embryo. (F) Limited-cycle RT-PCR analysis performed on RNA isolated from wild-type and Chx10orJ/orJ whole eyes at E13.5. Primers for A-, J-, H-, D- and M-Mitf are as used for Fig. 3A. For pan-specific amplification, primers in exon 5 and 7 were used. Note that for A- and J-Mitf, at the lower number of PCR cycles the intensities of the bands are slightly increased in mutant compared with wild-type eyes, but at the higher number of cycles the difference is no longer visible. No such differences are seen for M-Mitf. H- and D-Mitf, however, show a clear difference between wild type and mutant at both 29 and 30 cycles of amplification. The use of primers specific for cyclin D1 indicates a reduction in mutant, consistent with the corresponding retinal hypoproliferation.

 

Figure 7
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  		Fig. 7. The Mitfmi-rw allele partially rescues the Chx10orJ mutant phenotype. Eyes from newborn mice of the indicated genotypes were sectioned and processed for in situ hybridization with a pan-Mitf probe (A-D), cyclin D1 immunofluorescence (E-H), or PAX6/TUJ1 double immunofluorescence (I-L). Compared with wild-type (wt), Mitfmi-rw/mi-rw retinas appear normal, both in thickness and in staining. By contrast, Chx10orJ/orJ retinas retain Mitf expression and are severly hypoplastic, with a pigmented monolayer replacing the retina particularly in the distal part (B). Moreover, they show fewer cyclin D1-positive and PAX6-positive cells (F,J). Eyes from Mitfmi-rw/mi-rw;Chx10orJ/orJ double mutants, however, have retinas of relatively normal appearance and thickness even though their PAX6 staining and lamination are still abnormal (C,G,K). Brackets at the bottom mark the thickness of the retina.

 

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© The Company of Biologists Ltd 2008