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Sensory organ remodeling in Caenorhabditis elegans requires the zinc-finger protein ZTF-16.

Procko C, Lu Y, Shaham S - Genetics (2012)

Bottom Line: We show that ztf-16 mutants exhibit pronounced remodeling defects, which are explained, at least in part, by defects in the expression of ver-1.Expression and cell-specific rescue studies suggest that ztf-16, like ttx-1, functions within glia; however, promoter deletion studies show that ztf-16 acts through a site on the ver-1 promoter that is independent of ttx-1.Our studies identify an important component of glia remodeling and suggest that transcriptional changes may underlie glial morphological plasticity in the sensory organs of C. elegans.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Developmental Genetics, The Rockefeller University, New York, New York 10065, USA.

ABSTRACT
Neurons and glia display remarkable morphological plasticity, and remodeling of glia may facilitate neuronal shape changes. The molecular basis and control of glial shape changes is not well understood. In response to environmental stress, the nematode Caenorhabditis elegans enters an alternative developmental state, called dauer, in which glia and neurons of the amphid sensory organ remodel. Here, we describe a genetic screen aimed at identifying genes required for amphid glia remodeling. We previously demonstrated that remodeling requires the Otx-type transcription factor TTX-1 and its direct target, the receptor tyrosine kinase gene ver-1. We now find that the hunchback/Ikaros-like C2H2 zinc-finger factor ztf-16 is also required. We show that ztf-16 mutants exhibit pronounced remodeling defects, which are explained, at least in part, by defects in the expression of ver-1. Expression and cell-specific rescue studies suggest that ztf-16, like ttx-1, functions within glia; however, promoter deletion studies show that ztf-16 acts through a site on the ver-1 promoter that is independent of ttx-1. Our studies identify an important component of glia remodeling and suggest that transcriptional changes may underlie glial morphological plasticity in the sensory organs of C. elegans.

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ZTF-16 regulates expression from the ver-1 promoter through a site independent of the TTX-1-binding site. The indicated ver-1 promoter fragments (left, boxes) were fused to a gfp fluorescent reporter and tested for expression in the AMsh glia in adults raised at 15° and 25° and in dauers induced by starvation at 15° and 25°. The ver-1 gene fragments used are indicated relative to the +1 start codon. The ver-1 promoter::gfp transgene used for the mutant screen (nsIs22) is shown at the top (∼2 kb of upstream promoter sequence through +263 of the ver-1 gene). The positions of the TTX-1-binding site (Procko et al. 2011) and the Hunchback (Hb)-related binding site are shown. The asterisk indicates a mutated site (see Results). To test if ztf-16 was required for expression of a particular reporter, the reporter was crossed to ztf-16(ns171) mutants. P-values of reporter expression in the ztf-16 mutant dauers at 25° were determined by comparing the mutant strain against wild type using a χ2 test. The integrated transgenes and extrachromosomal arrays used were, from top to bottom, nsIs22, nsEx1136, nsEx2174, nsEx1269, and nsEx3022. Arrays are representative of others. n.d., not determined; n.s., not significant.
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fig8: ZTF-16 regulates expression from the ver-1 promoter through a site independent of the TTX-1-binding site. The indicated ver-1 promoter fragments (left, boxes) were fused to a gfp fluorescent reporter and tested for expression in the AMsh glia in adults raised at 15° and 25° and in dauers induced by starvation at 15° and 25°. The ver-1 gene fragments used are indicated relative to the +1 start codon. The ver-1 promoter::gfp transgene used for the mutant screen (nsIs22) is shown at the top (∼2 kb of upstream promoter sequence through +263 of the ver-1 gene). The positions of the TTX-1-binding site (Procko et al. 2011) and the Hunchback (Hb)-related binding site are shown. The asterisk indicates a mutated site (see Results). To test if ztf-16 was required for expression of a particular reporter, the reporter was crossed to ztf-16(ns171) mutants. P-values of reporter expression in the ztf-16 mutant dauers at 25° were determined by comparing the mutant strain against wild type using a χ2 test. The integrated transgenes and extrachromosomal arrays used were, from top to bottom, nsIs22, nsEx1136, nsEx2174, nsEx1269, and nsEx3022. Arrays are representative of others. n.d., not determined; n.s., not significant.

Mentions: We previously showed that robust expression of ver-1 promoter::gfp transgenes requires residues +1 to +263 of the ver-1 gene (relative to the ATG start site). We further described a smaller ∼90-bp interval sufficient for weak expression of the reporter in glia upon dauer entry. Within this interval, we identified a direct TTX-1-binding site with the core-binding residues located between position +176 and +179 (Procko et al. 2011) (Figure 8). Strikingly, we find that the ztf-16(ns171) mutation reduces ver-1 reporter expression only if residues +221 to +263 of the ver-1 gene are present. Specifically, GFP expression in animals carrying a transgene in which residues +221 to +263 of the ver-1 promoter are deleted is not altered in ztf-16 mutants (compare expression in dauers at 25° in wild-type and ztf-16 animals carrying either the top construct or the second construct from the bottom in Figure 8). Within the region of ver-1 regulated by ztf-16, we identified a potential ZTF-16-binding site, CATGAAAAC, at positions +217 to +225 on the basis of homology to Drosophila Hunchback, which binds the consensus sequence (G/C)(C/A)TAAAAAA (Stanojevic et al. 1989). Mutating these residues to GGGCCCAAC resulted in reduced ver-1 promoter::gfp expression (compare expression from top and bottom constructs in wild-type adult animals at 25° in Figure 8), raising the possibility that ZTF-16 may bind directly to the ver-1 gene to regulate its expression. To test for direct binding in vitro, we initially attempted to purify soluble full-length GST::ZTF-16a or GST::ZTF-16b protein induced in E. coli, but were unable to do so. We were able to purify zinc fingers 2–6 of the protein, but these showed only weak, nonspecific binding to a 40-bp biotin-labeled probe from the ver-1 gene (data not shown). Thus, it remains unclear whether ZTF-16 directly binds ver-1.


Sensory organ remodeling in Caenorhabditis elegans requires the zinc-finger protein ZTF-16.

Procko C, Lu Y, Shaham S - Genetics (2012)

ZTF-16 regulates expression from the ver-1 promoter through a site independent of the TTX-1-binding site. The indicated ver-1 promoter fragments (left, boxes) were fused to a gfp fluorescent reporter and tested for expression in the AMsh glia in adults raised at 15° and 25° and in dauers induced by starvation at 15° and 25°. The ver-1 gene fragments used are indicated relative to the +1 start codon. The ver-1 promoter::gfp transgene used for the mutant screen (nsIs22) is shown at the top (∼2 kb of upstream promoter sequence through +263 of the ver-1 gene). The positions of the TTX-1-binding site (Procko et al. 2011) and the Hunchback (Hb)-related binding site are shown. The asterisk indicates a mutated site (see Results). To test if ztf-16 was required for expression of a particular reporter, the reporter was crossed to ztf-16(ns171) mutants. P-values of reporter expression in the ztf-16 mutant dauers at 25° were determined by comparing the mutant strain against wild type using a χ2 test. The integrated transgenes and extrachromosomal arrays used were, from top to bottom, nsIs22, nsEx1136, nsEx2174, nsEx1269, and nsEx3022. Arrays are representative of others. n.d., not determined; n.s., not significant.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig8: ZTF-16 regulates expression from the ver-1 promoter through a site independent of the TTX-1-binding site. The indicated ver-1 promoter fragments (left, boxes) were fused to a gfp fluorescent reporter and tested for expression in the AMsh glia in adults raised at 15° and 25° and in dauers induced by starvation at 15° and 25°. The ver-1 gene fragments used are indicated relative to the +1 start codon. The ver-1 promoter::gfp transgene used for the mutant screen (nsIs22) is shown at the top (∼2 kb of upstream promoter sequence through +263 of the ver-1 gene). The positions of the TTX-1-binding site (Procko et al. 2011) and the Hunchback (Hb)-related binding site are shown. The asterisk indicates a mutated site (see Results). To test if ztf-16 was required for expression of a particular reporter, the reporter was crossed to ztf-16(ns171) mutants. P-values of reporter expression in the ztf-16 mutant dauers at 25° were determined by comparing the mutant strain against wild type using a χ2 test. The integrated transgenes and extrachromosomal arrays used were, from top to bottom, nsIs22, nsEx1136, nsEx2174, nsEx1269, and nsEx3022. Arrays are representative of others. n.d., not determined; n.s., not significant.
Mentions: We previously showed that robust expression of ver-1 promoter::gfp transgenes requires residues +1 to +263 of the ver-1 gene (relative to the ATG start site). We further described a smaller ∼90-bp interval sufficient for weak expression of the reporter in glia upon dauer entry. Within this interval, we identified a direct TTX-1-binding site with the core-binding residues located between position +176 and +179 (Procko et al. 2011) (Figure 8). Strikingly, we find that the ztf-16(ns171) mutation reduces ver-1 reporter expression only if residues +221 to +263 of the ver-1 gene are present. Specifically, GFP expression in animals carrying a transgene in which residues +221 to +263 of the ver-1 promoter are deleted is not altered in ztf-16 mutants (compare expression in dauers at 25° in wild-type and ztf-16 animals carrying either the top construct or the second construct from the bottom in Figure 8). Within the region of ver-1 regulated by ztf-16, we identified a potential ZTF-16-binding site, CATGAAAAC, at positions +217 to +225 on the basis of homology to Drosophila Hunchback, which binds the consensus sequence (G/C)(C/A)TAAAAAA (Stanojevic et al. 1989). Mutating these residues to GGGCCCAAC resulted in reduced ver-1 promoter::gfp expression (compare expression from top and bottom constructs in wild-type adult animals at 25° in Figure 8), raising the possibility that ZTF-16 may bind directly to the ver-1 gene to regulate its expression. To test for direct binding in vitro, we initially attempted to purify soluble full-length GST::ZTF-16a or GST::ZTF-16b protein induced in E. coli, but were unable to do so. We were able to purify zinc fingers 2–6 of the protein, but these showed only weak, nonspecific binding to a 40-bp biotin-labeled probe from the ver-1 gene (data not shown). Thus, it remains unclear whether ZTF-16 directly binds ver-1.

Bottom Line: We show that ztf-16 mutants exhibit pronounced remodeling defects, which are explained, at least in part, by defects in the expression of ver-1.Expression and cell-specific rescue studies suggest that ztf-16, like ttx-1, functions within glia; however, promoter deletion studies show that ztf-16 acts through a site on the ver-1 promoter that is independent of ttx-1.Our studies identify an important component of glia remodeling and suggest that transcriptional changes may underlie glial morphological plasticity in the sensory organs of C. elegans.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Developmental Genetics, The Rockefeller University, New York, New York 10065, USA.

ABSTRACT
Neurons and glia display remarkable morphological plasticity, and remodeling of glia may facilitate neuronal shape changes. The molecular basis and control of glial shape changes is not well understood. In response to environmental stress, the nematode Caenorhabditis elegans enters an alternative developmental state, called dauer, in which glia and neurons of the amphid sensory organ remodel. Here, we describe a genetic screen aimed at identifying genes required for amphid glia remodeling. We previously demonstrated that remodeling requires the Otx-type transcription factor TTX-1 and its direct target, the receptor tyrosine kinase gene ver-1. We now find that the hunchback/Ikaros-like C2H2 zinc-finger factor ztf-16 is also required. We show that ztf-16 mutants exhibit pronounced remodeling defects, which are explained, at least in part, by defects in the expression of ver-1. Expression and cell-specific rescue studies suggest that ztf-16, like ttx-1, functions within glia; however, promoter deletion studies show that ztf-16 acts through a site on the ver-1 promoter that is independent of ttx-1. Our studies identify an important component of glia remodeling and suggest that transcriptional changes may underlie glial morphological plasticity in the sensory organs of C. elegans.

Show MeSH
Related in: MedlinePlus