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doi: 10.1242/10.1242/dev.00571

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C. elegans ZAG-1, a Zn-finger-homeodomain protein, regulates axonal development and neuronal differentiation

Scott G. Clark* and Catherine Chiu

Molecular Neurobiology Program, Department of Pharmacology, Skirball Institute, NYU School of Medicine, New York, NY 10016, USA



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  		Fig. 1. (A) Wild-type, sra-6::gfp adult hermaphrodite. (B) zag-1(zd85); sra-6::gfp; arrow indicates position of PVQ. (C) Wild-type tph-1::gfp. (D) zag-1(zd85) tph-1::gfp; arrow indicates position of HSN. (E) Wild type tph-1::gfp. (F,G) zag-1(zd86); odr-2::cfp; arrows indicate abnormal axons. Although not visible in these images, HSN axons in zag-1 animals typically enter the ventral nerve cord. Anterior is leftwards; dorsal is towards the top.

 


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  		Fig. 2. zag-1 mutations block formation of stereotypic axon branches and cause pathfinding defects. Wild-type (A,C,E,G) and zag-1 (B,D,F,H) GFP-transgenic animals. Neurons: ADE (A,B); PDE (C,D); PVC (E,F) (additional GFP-labeled axons present in VNC); ALM and AVM (G,H). (H) AVM often terminates at the nerve ring in zag-1 mutants, although in this image AVM is wild type. Arrows indicate locations of missing axon branches and asterisks indicate ectopic glr-1::gfp-expressing neurons. (I) Schematic of wild-type and zag-1 axonal structures, not to scale. Anterior is leftwards (A,B,E,F,I), anterior is rightwards (C,D,G,H), dorsal is towards the top.

 


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  		Fig. 3. Genetic and molecular analysis of zag-1. (A) Genetic map of zag-1 IV region. (B) Schematic of cosmid F28F9 and subclones. Arrows depict hypothetical gene transcripts and direction of transcription (www.wormbase.org) (+, zag-1 rescue; -, no rescue). Restriction site abbreviations are K (KpnI), P (PstI) and S (SalI). (C) C. elegans and C. briggsae zag-1 genomic structures derived from cDNA and genomic sequences. C. briggsae zag-1 structure is hypothetical and based on a comparison with C. elegans sequences. Black boxes indicate exons, white boxes depict 3' UTR and vertical marks represent CACCT(G) motifs. VISTA server was used for some genomic sequence alignment (Mayor et al., 2000Go). (D) C. elegans ZAG-1 protein structure. Black boxes depict Zn-finger domains, the dark-gray box represents homeodomain and the light-gray box indicates PLDLT motif.

 


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  		Fig. 4. (A) Alignment of predicted amino acid sequences of ZAG-1 proteins in C. elegans (C. e.) and C. briggsae (C. b.). C. elegans sequence is based on full-length zag-1 cDNA (GenBank Accession Number AY224511). C. briggsae sequence is derived from the gene structure in Fig. 3C. Hyphens indicate gaps inserted to maintain alignment and periods represent identical residues in C. briggsae sequence. The homeodomain and five Zn-finger domains are boxed in gray and black, respectively. PLDLT sequence required for association with CtBP is underlined. Inverted carets mark the Q and W residues altered in zd85 and zd86 and arrows mark the relative positions of conserved splice sites. (B) Alignment of Zn-finger domain sequences from C. elegans ZAG-1, Drosophila ZFH-1, mouse {delta}EF1 and mouse SIP1. Modified, consensus C2H2-type Zn-finger sequence is shown on top ({psi}, aliphatic amino acid). (C) Alignment of homeodomain sequences from C. elegans ZAG-1, ZC123.3 (ZFH-2-like protein), MEC-3 and LIM-4.

 


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  		Fig. 5. (A) Schematic of zag-1 GFP translational and transcriptional transgenes. Black boxes depict zag-1 exons, gray boxes represent the GFP gene (coding region and synthetic introns) and white boxes indicate the 3' UTR of unc-54. For neuronal GFP expression: +++, high expression in many head and tail neurons, including head command interneurons, RME, PVT and PVQ; ++, high expression in similar set of neurons, excluding head command interneurons; +, low expression in five or fewer neurons in head. For muscle GFP expression: +, high expression in anal depressor and intestinal muscles; #, transient, high expression in body wall muscles; -, no muscle expression. For zag-1(lf) derepression: +, high expression in command interneurons and VNC motor neurons; -, no expression in command interneurons and VNC motor neurons. Regions required for downregulation of zag-1 expression, muscle expression, neuronal expression and ZAG-1-mediated repression are indicated. The caret indicates the location of tandem CACCT site. (B) Alignment of CACCT and adjacent sequences present in C. elegans (C. e.) and C. briggsae (C. b.) zag-1 genomic region. Nucleotides altered in promoter mutants are indicated above the sequence.

 


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  		Fig. 6. zag-1 expression pattern. DIC (A) and fluorescence (B) image of zag-1::ZAG-1::GFP embryo with GFP expression in nerve ring nuclei (int., intestinal autofluorescence). (C) zag-1::gfp(zdIs21) (2.9 kb upstream fragment) adult hermaphrodite with expression in subset of nerve ring neurons, no expression in ventral nerve cord neurons. (D) Tail region of zag-1::gfp(zdIs21); zag-1(-918A,-895T)::rfp(zdIs41) adult. zag-1 promoter (4.0 kb) containing mutated CACCT sites directed RFP expression in more neurons than the wild-type promoter (GFP). Both promoters directed expression in anal depressor and intestinal muscles. (E) zag-1(zd86) zag-1::gfp(zdIs21). zag-1 mutations induced expression in ventral cord motoneurons. (F) zag-1(-918A,-895T)::rfp(zdIs41). zag-1 promoter with mutated CACCT sites directed RFP expression in ventral cord motoneurons. Anterior is leftwards (C), anterior is rightwards (D-F), dorsal is towards the top.

 

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