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. 2018 Jan 4;8(1):17-26.
doi: 10.1534/g3.117.300132.

A Caenorhabditis elegans Zinc Finger Transcription Factor, ztf-6, Required for the Specification of a Dopamine Neuron-Producing Lineage

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A Caenorhabditis elegans Zinc Finger Transcription Factor, ztf-6, Required for the Specification of a Dopamine Neuron-Producing Lineage

Maria Doitsidou et al. G3 (Bethesda). .
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Abstract

Invertebrate and vertebrate nervous systems generate different types of dopaminergic neurons in distinct parts of the brain. We have taken a genetic approach to understand how the four functionally related, but lineally unrelated, classes of dopaminergic neurons of the nematode Caenorhabditis elegans, located in distinct parts of its nervous system, are specified. We have identified several genes involved in the generation of a specific dopaminergic neuron type that is generated from the so-called postdeirid lineage, called PDE. Apart from classic proneural genes and components of the mediator complex, we identified a novel, previously uncharacterized zinc finger transcription factor, ztf-6 Loss of ztf-6 has distinct effects in different dopamine neuron-producing neuronal lineages. In the postdeirid lineage, ztf-6 is required for proper cell division patterns and the proper distribution of a critical cell fate determinant, the POP-1/TCF-like transcription factor.

Keywords: PDE; Zing finger transcription factor dopaminergic neurons; mediator complex lineage analysis; mutant screen report; postdeirid lineage C. elegans; ztf-6.

Figures

Figure 1
Figure 1
Dopaminergic neurons of the C. elegans hermaphrodite and their lineages. Lineage data comes from (Sulston and Horvitz 1977; Sulston et al. 1983). Dopaminergic neurons were first identified by Sulston et al. (1975). ADE, anterior deirid sensillum; CEP, cephalic sensillum; CEPD, dorsal CEP; CEPV, ventral CEP; PDE, postdeirid sensillum.
Figure 2
Figure 2
ztf-6(ot271) mutant animals show defects in lineages that produce dopaminergic neurons. (A) Expression of the dopamine transporter dat-1::gfp (vtIs1) in wild-type and ztf-6 mutant backgrounds. Yellow arrows indicate dopaminergic cell bodies normally present, red arrows indicate extra cells generated, and the dotted red circles expected positions of missing cells. (B) Quantification of defects. “1” indicates cells located in close proximity to the original neuron. (C) Terminal markers fail to be expressed in the postdeirid lineage of ztf-6 mutants. (D) Seam cell defects in ztf-6 mutants. Seam cells are shown in early larval stages when V cells divide. (E) Characteristic cluster of neuro- and glioblast cells of the postdeirid lineage, observed in wild-type animals. These clusters are difficult to observe in ztf-6 mutants. ADE, anterior deirid sensillum (ADE); CEP, cephalic sensillum; CEPD, dorsal CEP; CEPV, ventral CEP; PDE, postdeirid sensillum.
Figure 3
Figure 3
Lineage analysis of the postdeirid of ztf-6 mutants. Black bar, 10 μm in all panels. Presence of large hypodermal nuclei not related to the postdeirid or L2 seam cell lineage are designated as “hyp” in the initial image and circled with a black dashed line. The unilateral SDQL and PVM neurons are circled in a dashed white circle, when present, to differentiate them from cells in the postdeirid lineage. (A–C) Examples of V5.p lineages in wild-type and ztf-6 animals. Nuclear marker expression (stIs10166) in the V5.p lineage was followed over time to determine cell division times and postdeirid phenotypic information. Cell colors in the lineage chart correspond to colored arrows and cells in the images. Postdeirid lineages in older animals with faded nuclear marker expression are circled in a solid white line, due to the loss of expression of the hypodermal fate marker later in development. If present in the genetic background, vtIs1 (dat-1::gfp) is shown overlaid in green. (D) Temporal defect in postdeirid lineage formation in a ztf-6(ot271) mutant. An ectopic postdeirid lineage contained by a gray outline was already present at the start of the L2 stage before data acquisition was initiated. During the L2 stage, V5.p produced a postdeirid lineage, resulting in two dat-1::gfp expressing neurons. dat-1::gfp expression turned on at similar times for both postdeirid sensillum (PDE) neurons in the late L2 stage. (E) Spatial patterning defects in two different ztf-6(ot271) animals showing spurious cell divisions in the V4.p lineages. Ectopic dat-1::gfp expressing cells appear in the V4 region of both animals due to increased cell division. (F) Late L2 stage wild-type and ztf-6(ot271) animals showing reversed asymmetry or lack of asymmetry of dpy-7 expression in V5.p mutant lineages. Near the end of the L2 stage (10–14 hr after start of L2 stage), the remaining cells in the V5.p lineage were scored for strong dpy-7 expression as a proxy for hypodermal fate, with the D notation representing the presumptive differentiated (hypodermal) cell fate based on relative expression level between seam cells and existing hypodermal nuclei. Wild-type image directly from (A) 680’ time point, showing the proper dpy-7/differentiation pattern of the V5.ppa and V5.ppp cells. Mutant animal (i) shows lack of asymmetry, with neither seam cell taking the hypodermal fate, while mutant animal (ii) shows reversed asymmetry. Some seam cells are circled with a dashed yellow line for easier visualization. (G) Late L2 stage wild-type and ztf-6(ot271) mutant V4.p lineages showing dpy-7 expression patterns. In the wild-type animal, expression levels in the V4.paa and V4.ppa cells indicate the hypodermal fate, marked with a D (differentiated). In the mutant, all of the V4 seam cells acquired the hypodermal fate instead of only the anterior of each pair. Also, note the abnormally clumped spacing of the nuclei in the mutant.
Figure 4
Figure 4
ztf-6 affects asymmetric POP-1 localization. (A) Schematic of the V5.p asymmetric divisions before specification of the postdeirid lineage. The anterior and posterior sister cell pairs resulting from the division of V5.pa and V5.pp were analyzed for asymmetric POP-1 localization in the mid-L2 stage. POP-1::GFP levels were quantified and the asymmetric distribution was determined in wild-type and ztf-6 mutant animals. Black bar, 12 μm. (B) Comparison of the distribution of POP-1::GFP in V5.pa and V5.pp daughter cells. There is an increase in sister cells pairs with inverted POP-1 localization (A < P) in the mutant compared to control (P < 0.003, Fisher Exact Test). (C) Comparison of the logarithm of the ratio of POP-1::GFP between sister cells. Each marker represents one sister cell pair, with black representing anterior pairs and gray representing posterior pairs (data plotted from (B)). Magenta dots in the plot correspond to the sister cell pairs shown in (A). Dashed black lines indicate the ratios between which we considered the sister cells pairs symmetrical. GFP, green fluorescent protein.
Figure 5
Figure 5
Cloning and expression of ztf-6. (A) Summary of whole-genome sequence data of ztf-6(ot280). (B) ztf-6 gene locus, reporter gene used in this study, and ZTF-6 protein structure. Nucleotide changes are as follows: ot271 and ot273, 5′-TGGCAGGAAGTGGTGTTGGAGTTGCCGAACGGCT-3′ (underlined: 16 bp insertion); ot274, C to T in 5′-ATGGTCTTCGATTGCATAGA-3′; and ot280, G to A in 5′-AGAAATCCAGCATGCATGGAAG-3′. (C) Expression of ztf-6::gfp (otEx6298) at the L1 and L2 stages. Note that some autofluorescence is visible as small bright dots in the middle part of the body. (D) ztf-6::gfp expression in the V5 lineage. Animals were staged by egg-laying and analysis at different stages, as indicated. EMS, Ethyl Methanesulfonate; SNP, single nucleotide polymorphism; VNC, ventral nerve cord.

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