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doi: 10.1242/10.1242/dev.00419

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Zic1 represses Math1 expression via interactions with the Math1 enhancer and modulation of Math1 autoregulation

Philip J. Ebert1, John R. Timmer2, Yuji Nakada1, Amy W. Helms1, Preeti B. Parab1, Ying Liu1, Thomas L. Hunsaker1 and Jane E. Johnson1,*

1 Center for Basic Neuroscience, University of Texas Southwestern Medical Center, Dallas, TX 75390-9111, USA
2 Developmental Biology Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA



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  		Fig. 5. Characterization of the role of the Zic1-binding site for enhancer activity using electroporation of chick neural tube. (A) The transgenic constructs used in this study (Tg#) and a summary of their activity (ß-gal exp) when electroporated into chick neural tube (EP chick). Each Math1 enhancer fragment was cloned 5' to the promoter region in the BGZA reporter. The numbers in the diagram relate to the published nucleotide sequence of the Math1 enhancer (Accession Number, AF218258). The specific mutations are shown in Fig. 1B. Representative data for each construct are shown in B as cross-sections of chick neural tubes after electroporation and X-gal staining. Electroporated side is always on the right. Arrows indicate expression of the transgene on the electroporated side.

 


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  		Fig. 1. Sequence and functional conservation between Math1 and Cath enhancers. (A) The percent sequence conservation between the Math1 enhancer (Accession Number, AF218258) and sequences in human (HATH1, Accession Number, AF18259) and chicken (Cath1, Accession Number, AF467292). These sequences are found 3' to the coding sequences, ~3 kb for Math1, ~3.4 kb for HATH1 and ~1.7 kb for Cath1. (B) Sequence from 1192 to 1260 comparing mouse, human and chick sequences across the region containing the essential E-box and the Zic1-binding site in the Math1 enhancer. Details of the mutations in the constructs shown in Fig. 5 are shown. (C) The Math1 enhancer (Math1/lacZ Tg) and (D) the Cath1 enhancer (Cath1/lacZ Tg) have the same activity in E10.5 transgenic embryos as shown with whole-mount X-gal stained embryos. (E) Cross-section of the neural tube of a Math1/lacZ transgenic mouse embryo showing the dorsal pattern of expression for comparison with F, which is the expression of the same transgene after electroporation into one side of a chick neural tube (arrow). Note the DNA enters cells along the DV axis but expression of the transgene is restricted dorsally.

 


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  		Fig. 3. A novel Zic1 site identified within the Math1 enhancer. Gel shift assays with in vitro transcribed and translated Zic1 (lacking the N-terminal 110 amino acids) and probes generated from (A) a 30 bp sequence from the Math1 enhancer containing the newly identified Zic1-binding site (Z-site wt), or (B) the wild-type 374 bp enhancer B (Math1 enh probe) and Math1 enh probes mutated in the Zic1 site (mutations shown in C). Sequence of the oligonucleotide probe and the cold competitor DNAs are shown in C. Control extract is the reticulocyte extract with no added template. Zic/Gli probe is the previously published Zic consensus binding site. (C) Nucleotide requirements for Zic1 binding defined by EMSA with mutant oligonucleotides are shown. The novel Zic1 site defined here (consensus) has little similarity to the published Zic binding site (Gli site).

 


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  		Fig. 2. Comparison of Math1 and Zic protein expression. Double immunofluorescence labelling with antisera against Zic factors (green) (A,C) and Math1 (red) (B,C) in cross-sections of mouse E10.5 dorsal neural tube. (D-F) Higher magnification images of the region comparable with the boxed region in C. The region of lowest Zic expression (A,D, arrows) is the region where Math1 is expressed in the dorsal neural tube (C,F). Note that the cells expressing highest levels of Math1 do not express Zic factors. Cells expressing low levels of both factors are detected (arrowheads). (G) Zic factor immunofluorescence in E10.5 Math1 mutant dorsal neural tube. Note the uniform Zic expression when compared with wild type (D). Scale bar: 100 µm in A-C; 35 µm in D-G.

 


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  		Fig. 4. Overexpression of Zic1 represses the expression of Cath1 and the Math1 enhancer in chick neural tubes. Control (A,C) and mycZic1 (B,D), both Myc tagged and in expression vector pMiWIII were electroporated into chick stage HH12-14 and assayed at 24 hours for endogenous levels of Cath1 (A,B) and for the Myc tag (A',B') by immunofluorescence. Arrowheads indicate the electroporated side of the neural tube. The graph shows the number of Cath1-expressing cells/section on the electroporated (gray bars) and non-electroporated (black bars) sides of the neural tube. At least three sections each from three embryos were counted for each construct. (C,D) Math1/lacZ transgene was co-electroporated with the different expression constructs into HH14-17 chick neural tubes and assayed at 24 hours by immunofluorescence for ß-gal (C,D) or Myc (C',D'). Scale bar: 50 µm.

 


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  		Fig. 6. Zic1 represses Math1/Cath1 autoregulation. Chick neural tubes electroporated with Math1 (A) or Math1 plus mycZic1 (C) at HH14 and assayed at 24 hours for Cath1 expression by mRNA in situ hybridization. Arrows indicate the electroporated side. Math1 activates Cath1 expression (A) but this activity is inhibited by mycZic1 (C). Chick neural tubes co-electroporated with a Math1/lacZ transgene plus Math1 and Myc-tagged control vector (B), or Math1 and mycZic1 (D). Immunofluorescence of adjacent sections to detect ß-gal (B,D), Math1 (B',D'), Myc (B''), or Zic (D'') are shown. Note the dramatic activation of the Math1/lacZ transgene when co-electroporated with Math1 (compare Fig. 3E or 5C with B) and the block of this activation in the presence of mycZic1 (D).

 


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  		Fig. 7. Math1 promotes cell movement out of the ventricular zone while Zic1 maintains cells within this zone, both at the expense of neural crest cells. Neural tubes of HH12 chick embryos were electroporated (right side as shown) with CMVpN1-EGFP alone (A,B), with Math1 (C,D) or with mycZic1 (E,F) expression constructs and harvested at 48 hours. Whole-mount GFP images (A,C,E) or transverse sections imaged for GFP (green) and islet1 (red) immunofluorescence (B,D,F) are shown. The islet1 staining allows visualization of the motor pools (MN) and the neural crest-derived dorsal root ganglia (DRG). Arrows in A,B indicate clear GFP in DRG on the electroporated side with lower levels in DRG on the non-electroporated side. Math1 and mycZic1 biases cells away from DRG as seen in the near absence of GFP in this structure (C-F). Within the neural tube, GFP in controls spans the width of the neural tube (A,B), whereas Math1 overexpressing cells are biased towards the lateral tube where differentiated cells reside (C,D) and mycZic1 overexpression biases cells to the ventricular side where progenitors reside (E,F). The asterisks indicate the location of the apical surface of the neural tube. Arrows in D,F indicate DRG.

 


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  		Fig. 8. Zic1 represses Cath1 and Cash1 but not chick Ngn1 and Ngn2. (A-D) Neural tubes of HH14 chick embryos were electroporated on the right side (arrowheads) with mycZic1 and harvested at 24 hours. mRNA in situ hybridization with Cath1 (A), Cash1 (B), Ngn1 (C) and Ngn2 (D) are shown. Relative to the control side (left) in each case, Cath1 and Cash1, but not Ngn1/2, are repressed by overexpression of mycZic1. (E-H) Double label immunofluorescence using anti-Mash1 (red) and anti-Zic (green) antisera on E11.5 mouse spinal neural tube. Yellow cells indicate overlap in expression in the dorsal Mash1 domain (F-H, arrowheads). Scale bar: 100 µm in A-E; 50 µm in F-H.

 

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