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First published online 4 April 2007
doi: 10.1242/dev.02845

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The proneural determinant MASH1 regulates forebrain Dlx1/2 expression through the I12b intergenic enhancer

Center for Advanced Research in Environmental Genomics (CAREG), Department of Biology, University of Ottawa, 20 Marie Curie, K1N 6N5, Ottawa, Ontario, Canada.

View larger version (69K): [in this window] [in a new window]   Fig. 1. The I12b intergenic enhancer targets the expression of a lacZ reporter construct to the developing mouse forebrain. (A) The convergently transcribed (arrows) mouse Dlx1 and Dlx2 genes. Exons are shown as blue boxes. Two enhancers, I12a and I12b, are located in the intergenic region. The reporter construct used for the production of mouse transgenic lines is shown beneath. (B) The I12b enhancer targets expression of the reporter gene to the ventral telencephalon (VT), the diencephalon (Di) and the frontonasal prominence (FN), in an E11.5 mouse embryo (whole-mount preparation). (C-F) Transverse sections (half a section is shown in E,F) through the telencephalon of E11.5 (C) and E13.5 (D) transgenic embryos and rostral brain of P0 (E) and P25 (F) transgenic mice. Scale bars: 1 mm in B; 500 µm in C-F.

View larger version (32K): [in this window] [in a new window]   Fig. 2. Trans-acting factors from E13.5 mouse embryonic brain bind to the I12b enhancer. (A) DNase I footprinting analysis of two overlapping DNA fragments of the I12b enhancer (nt 1-292 and 259-437) using a nuclear protein extract from E13.5 mouse forebrain. Six regions of the enhancer (FP1 to FP6) are bound by nuclear proteins. Protected areas (footprints) are represented by thick lines and hypersensitive sites by arrowheads. Numbers to the left of each panel indicate relative positions on the enhancer. (B) Protein-DNA interactions mapped (using GA Sequence in A) on the mouse I12b enhancer sequence. The protected areas identified by DNase I footprinting (FP1-FP6) are boxed. The two fragments used for the footprinting analysis are indicated by colored arrows (red arrows, nt 1 to 292; blue arrows, nt 259-437). Sequences of the human (H) and zebrafish (Z) I12b sequences are compared with mouse (M). Protein-DNA interactions are mostly located in regions with a high degree of sequence similarity. (C) Reporter constructs containing mutations (red) of the FP sites in the I12b enhancer.

View larger version (8K): [in this window] [in a new window]   Fig. 3. Dlx2a can bind and activate transcription through the intergenic I12b enhancer. (A) In a transient transfection assay, zebrafish Dlx2a upregulates transcription of a CAT reporter construct containing the I12b enhancer (mI12b-pBLCAT2) by a factor of 11. By comparison, expression of a CAT reporter gene under control of the I56i enhancer (Zerucha et al., 2000) was activated 5.6-fold in the presence of Dlx2a. (B) The disruption of the Dlx2a homeodomain completely abolishes this activation. In the presence of zebrafish Dlx2a, a 9.03-fold increase in CAT activity was observed, whereas the Dlx2a homeodomain mutant showed only a basal level of CAT activity. Values shown represent the mean of relative CAT activity obtained from two to three independent experiments ± s.e.m.

View larger version (79K): [in this window] [in a new window]   Fig. 4. Effect of mutations in FP sites on I12b enhancer activity. Lateral views of E11.5 transgenic mouse embryos showing expression of the lacZ reporter in the forebrain. Wild-type (I12b-lacZ) enhancer (A) and enhancers with mutagenized (B,E) DLX/MSX/NKX2.5 sites, (C) MEIS1 site, (D) FP3 homeodomain-binding site, (F) MASH1 site, and (G) FP6 homeodomain site. VT, ventral telencephalon; Di, diencephalon. FN, frontonasal prominence. Scale bar: 1 mm.

View larger version (45K): [in this window] [in a new window]   Fig. 5. Impaired I12b enhancer activity in the telencephalon following mutagenesis of protein-binding sites. Transverse sections through the telencephalon of E11.5 transgenic mouse embryos (such as those shown in Fig. 4). The plane of section is schematized. Two series of transverse sections corresponding to the rostral (A,C,E,G,I,K,M) and middle (B,D,F,H,J,L,N) regions of the telencephalon are shown. Expression obtained with the wild-type I12b enhancer (A,B) and following mutagenesis of (C,D) the first DLX/MSX/NKX2.5-binding site, (E,F) MEIS1 site, (G,H) FP3 site, (I,J) second DLX/MSX/NKX2.5 site, (K,L) MASH1 site, and (M,N) FP6 homeodomain site. Cx, cortex; IZ, intermediate zone; LGE, lateral ganglionic eminence; MGE, medial ganglionic eminence; AEP, anterior entopeduncular area; POA, preoptic area; VT, ventral telencephalon; SCB, suprachiasmatic band (a diencephalic structure). Scale bar: 500 µm.

View larger version (69K): [in this window] [in a new window]   Fig. 6. Mutagenesis of protein-binding sites on the I12b enhancer affects its activity in the diencephalon. Transverse sections through the diencephalon, at the caudal limit of the telencephalon, in E11.5 transgenic mouse embryos (such as those shown in Fig. 4). The plane of section is schematized. Activity of the wild-type I12b enhancer (A) and following mutagenesis of (B) the first DLX/MSX/NKX2.5 site, (C) MEIS1 site, (D) FP3 site, (E) second DLX/MSX/NKX2.5 site, (F) MASH1 site, and (G) FP6 site. Scale bar: 500 µm.

View larger version (45K): [in this window] [in a new window]   Fig. 7. The bHLH transcription factor MASH1 can bind and activate transcription through an E-box sequence in the I12b enhancer. (A) In a transient co-transfection assay, MASH1 activates transcription of a CAT reporter construct containing the I12b enhancer (mI12b-pBLCAT2) by a factor of about four. Values shown represent the mean relative CAT activity obtained from two independent experiments ± s.e.m. (B) EMSA using the FP5 sequence (see Fig. 2) shows binding to a combination of recombinant (r) MASH1 and E47 (lanes 2-4, arrowheads). This binding is achieved through an E-box present in the FP5 sequence. rMASH1 and rE47 were unable to bind FP5 in which the E-box was mutated (lanes 6-8; same mutation as for the transgenic mouse experiments). (C) Preincubation of rMASH1 and rE47 with an anti-MASH1 antibody completely abolishes the formation of the rMASH-rE47-FP5 complexes (lanes 3-5, arrowheads), in contrast to a binding reaction that was preincubated with the control antibody (lanes 7-9).

View larger version (27K): [in this window] [in a new window]   Fig. 8. MASH1 binds to the I12b enhancer in vivo. LGE and MGE were dissected from E11.5 mouse telencephalon, cross-linked and immunoprecipitated using an anti-MASH1 antibody (-Mash1). PCR analysis was performed on the immunoprecipitated chromatin using primers corresponding to the I12b enhancer (I12b Enh) and the Hes6 upstream enhancer (Hes6 Enh). After 28-30 cycles, PCR products (605 bp for mI12b and 176 bp for Hes6) were electrophoresed on 1% agarose gel. As controls, amplifications were carried out on chromatin obtained from the elution of protein-A sepharose beads that was preincubated with the cross-linked chromatin or with the IP dilution buffer (No antibody and Mock, respectively). Different dilutions of total input chromatin (Genomic DNA) were used for each set of primers: 1/500 for I12b and 1/1000 for Hes6 Enh.

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