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First published online 16 November 2005
doi: 10.1242/dev.02163

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A novel C. elegans zinc finger transcription factor, lsy-2, required for the cell type-specific expression of the lsy-6 microRNA

Department of Biochemistry and Molecular Biophysics, Center for Neurobiology and Behavior, Howard Hughes Medical Institute, Columbia University Medical Center, 701 West 168th Street, New York, NY 10032, USA

View larger version (63K): [in a new window]   Fig. 1. The ASER versus ASEL fate decision in wild-type and lsy-2 mutant animals. (A) Schematic representation of the bilaterally symmetric ASE gustatory neurons. Their bilaterality extends to cell position, axonal and dendritic morphology, synaptic connectivity (White et al., 1986) and the expression of a large number of bilaterally expressed genes (www.wormbase.org). The enlarged images illustrate the fate differences between ASEL and ASER, and provide a summary of the genetic regulatory network that controls the ASEL and ASER fates (Chang et al., 2004; Chang et al., 2003; Hobert et al., 1999; Johnston and Hobert, 2003; Johnston et al., 2005). The permissively acting factors lin-49, unc-37 and ceh-36 are not shown but are referred to in the Discussion. (B) In lsy-2 mutant animals, ASEL-specific expression of gcy-7, assayed using a gcy-7prom::gfp transgene (otIs3), is lost and ASER-specific expression of gcy-5, assayed with a gcy-5prom::gfp transgene (ntIs1), is derepressed in ASEL. lsy-2(ot64) null mutant animals are shown. See Table 1 for quantification of the data and more alleles.

View larger version (23K): [in a new window]   Fig. 2. Mapping of the lsy-2 locus. (Top) A genetic map (not drawn to scale); (bottom) a physical map. lsy-2 was mapped with the SNP markers pkP6142 and pkP6144 in the Hawaiian C. elegans isolate CB4856 (Hodgkin and Doniach, 1997; Wicks et al., 2001). An unc-1 lsy-2 dpy-3 triple mutant was generated and used to three-factor map lsy-2 between pkP6143 (located on cosmid F49H12) and dpy-3 (EGAP7.1). The blue dots in the schematic presentation of the LSY-2 protein (not drawn to scale) indicate Cys- and Hischelated zinc atoms; the numbers indicate the spacing between individual Cys and His residues. NLS, putative nuclear localization sequence.

View larger version (55K): [in a new window]   Fig. 3. lsy-2 encodes a C2H2 zinc finger protein. (A) Sequence alignment of lsy-2 and its paralog, lsl-1, and their C. briggsae orthologs. Conserved cysteines and histidines in the C2H2 fingers (grey boxes) are indicated in red. A putative nuclear localization sequence is underlined. Mutant alleles are indicated in blue. (B) Alignment of the first three C2H2 fingers of LSY-2 with representative members of the SP1/KLF-like family (Kaczynski et al., 2003) and two other SP1/KLF-like C. elegans proteins. These two proteins are the top hits when BLAST searching the C. elegans genome for human or Drosophila SP1-like proteins. Other KLF-like proteins have previously been noted in worms (Oates et al., 2001) and are not shown here. Residues in red and blue are 100% conserved; residues in green are partly conserved. Sequences of human proteins are from Kaczynski et al. (Kaczynski et al., 2003). (C) The linkers between C2H2 zinc fingers in the triple motif in LSY-2 have a similar length and share conserved amino acids with known transcription factors of the SP1/KLF family. One representative member of each of the three subgroups of human SP1/KLF proteins is shown.

View larger version (46K): [in a new window]   Fig. 4. lsy-2 is expressed ubiquitously and localizes to moving speckles in the nucleus. (A) lsy-2prom::gfp and lsy-2::gfp reporter gene constructs used in this study. The gray bar behind the green gfp sequence indicates the heterologous unc-54 3'UTR. (B) lsy-2prom::gfp and lsy-2::gfp reporter gene constructs are ubiquitously expressed at different developmental stages. Nine independent lsy-2::gfp transgenic lines and three independent lsy-2prom::gfp lines show similar expression patterns. (C) lsy-2::gfp is expressed in ASEL and ASER. ASEL and ASER are labeled with the ceh-36prom::rfp transgene otIs151. Note that the rfp reporter is diffusely localized throughout the cytoplasm, whereas the gfp signal is in nuclear speckles. lsy-2::gfp rescues the lsy-2 mutant phenotype (Table 3). (D) lsy-2::gfp is localized to moving nuclear speckles. Individual time frames of a movie, shot with Openlab Software at a time interval of half a second per frame (20 frames total), are displayed. The movie is available upon request.

View larger version (15K): [in a new window]   Fig. 5. lsy-2 is required for proper expression of feedback loop regulators. (A-C) No exp., no expression in ASEL and ASER; L>R, greater expression in ASEL versus ASER; L>0, expression in ASEL only; L=R, equal expression in ASEL and ASER. (A) Expression of lsy-6, assayed with the lsy-6prom::gfp transgene otIs160, is lost in the ASEL neuron of lsy-2(ot64) null mutant animals. (B) ASER-specific expression of cog-1, assayed with the cog-1::gfp transgene syIs63, is de-repressed in ASEL in lsy-2(ot64) null mutant animals. (C) mir-273 mediated downregulation of the die-1 3'UTR in ASER, assayed with the ceh-36prom::gfp::die-13'UTR transgene otEx1759, is disrupted in lsy-2(ot64) null mutant animals. (D) Placement of lsy-2 relative to the previously described bistable feedback loop in ASEL (left) and ASER (right). For a more detailed explanation of the regulatory interactions, see Johnston et al. (Johnston et al., 2005).