A Single Gene Target of an ETS-Family Transcription Factor Determines Neuronal CO2-Chemosensitivity

Many animals possess neurons specialized for the detection of carbon dioxide (CO2), which acts as a cue to elicit behavioral responses and is also an internally generated product of respiration that regulates animal physiology. In many organisms how such neurons detect CO2 is poorly understood. We report here a mechanism that endows C. elegans neurons with the ability to detect CO2. The ETS-5 transcription factor is necessary for the specification of CO2-sensing BAG neurons. Expression of a single ETS-5 target gene, gcy-9, which encodes a receptor-type guanylate cyclase, is sufficient to bypass a requirement for ets-5 in CO2-detection and transforms neurons into CO2-sensing neurons. Because ETS-5 and GCY-9 are members of gene families that are conserved between nematodes and vertebrates, a similar mechanism might act in the specification of CO2-sensing neurons in other phyla

EGL-13/SoxD Specifies Distinct O₂ and CO₂ Sensory Neuron Fates in <i>Caenorhabditis elegans</i>

<div><p></p><p>Animals harbor specialized neuronal systems that are used for sensing and coordinating responses to changes in oxygen (O₂) and carbon dioxide (CO₂). In <i>Caenorhabditis elegans</i>, the O₂/CO₂ sensory system comprises functionally and morphologically distinct sensory neurons that mediate rapid behavioral responses to exquisite changes in O₂ or CO₂ levels via different sensory receptors. How the diversification of the O₂- and CO₂-sensing neurons is established is poorly understood. We show here that the molecular identity of both the BAG (O₂/CO₂-sensing) and the URX (O₂-sensing) neurons is controlled by the phylogenetically conserved SoxD transcription factor homolog EGL-13. <i>egl-13</i> mutant animals fail to fully express the distinct terminal gene batteries of the BAG and URX neurons and, as such, are unable to mount behavioral responses to changes in O₂ and CO₂. We found that the expression of <i>egl-13</i> is regulated in the BAG and URX neurons by two conserved transcription factors—ETS-5(Ets factor) in the BAG neurons and AHR-1(bHLH factor) in the URX neurons. In addition, we found that EGL-13 acts in partially parallel pathways with both ETS-5 and AHR-1 to direct BAG and URX neuronal fate respectively. Finally, we found that EGL-13 is sufficient to induce O₂- and CO₂-sensing cell fates in some cellular contexts. Thus, the same core regulatory factor, <i>egl-13</i>, is required and sufficient to specify the distinct fates of O₂- and CO₂-sensing neurons in <i>C. elegans</i>. These findings extend our understanding of mechanisms of neuronal diversification and the regulation of molecular factors that may be conserved in higher organisms.</p></div

An ETS-family transcription factor is required for the specification of <i>C. elegans</i> CO₂-chemosensitive BAG neurons.

<p>(A) A 31 basepair DNA element comprising a single ETS-binding motif (top) drives expression of GFP specifically in the BAG chemosensitive neurons (bottom). (B) One of ten ETS-family transcription factors encoded by the <i>C. elegans</i> genome is required for specification of BAG neurons. Shown is percent of animals mutant for each of ten ETS-family transcription factors encoded by the <i>C. elegans</i> genome that are BAGL/R ON (green circles) and BAGL/R OFF (open circles) for expression of a <i>Prom_flp-19::gfp</i> reporter transgene. <i>N</i> = number of animals scored. # We found one <i>lin-1(e1777)</i> mutant in which <i>Prom_flp-19::gfp</i> was not expressed in BAGR. (C) Fluorescence micrographs of <i>Prom_flp-19::gfp</i> expression in a wild-type animal, an <i>ets-5</i> mutant and an <i>ets-5</i> mutant carrying a wild-type copy of the <i>ets-5</i> locus in a fosmid-derived transgene. BAGL/R neuron positions are marked by red circles and cells previously identified as AWAL/R <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034014#pone.0034014-Kim1" target="_blank">[19]</a> are marked by blue circles. The nerve ring is indicated by an arrowhead. The scale bar in lower panel is 20 µm. A: anterior, L: left. The <i>ets-5</i> mutant allele was <i>tm1734</i>. The <i>Prom_flp-19::gfp</i> transgene was <i>ynIs34</i> and the <i>ets-5</i> rescuing transgene was <i>rpEx246</i>.</p

ETS-5 directly interacts with the <i>gcy-9</i> promoter.

<p>(A) ETS-5::GFP associates with the <i>gcy-9</i> promoter <i>in vivo</i>. Anti-GFP immunoprecipitates were prepared from cross-linked extracts of wild-type animals or animals carrying a functional <i>ets-5::gfp</i> transgene and interrogated for the presence of <i>gcy-9</i> promoter sequences by PCR. Immunoprecipitates from transgenic animals were enriched for <i>gcy-9</i> promoter sequences that contained the ETS-binding site at −202 bp. Control sequences at −5000 bp were not enriched in immunoprecipitates from transgenic animals. The <i>ets-5::gfp</i> transgene used was <i>wzIs80</i>. (B) ETS-5 binds to <i>gcy-9</i> promoter sequences <i>in vitro</i>. A mobility shift assay was performed with recombinant GST::ETS-5 and a 45 bp biotinylated DNA duplex probe containing the ETS-binding site from the <i>gcy-9</i> promoter. Recombinant GST::ETS-5 but not GST alone altered the electrophoretic mobility of the probe. The interaction between GST::ETS-5 and the probe was blocked by a molar excess of unlabeled probe but not by an excess of scrambled probe with the same nucleotide composition. Excess unlabeled wild-type and scrambled competitor probe was added in the following molar ratios: 10×, 50×, 100×, 500×.</p

Expression of the ETS-5 target gene <i>gcy-9</i> restores CO₂-chemosensitivity to <i>ets-5</i> mutants.

<p>(A) <i>gcy-36</i> and <i>gcy-18</i> promoters, which are active in oxygen-sensing and thermosensory neurons, respectively, are not regulated by ETS-5. Shown are lateral views of wild-type and <i>ets-5</i> mutant animals carrying either a <i>gcy-36</i> reporter, which is expressed by the oxygen-sensing URX neurons, or a <i>gcy-18</i> reporter, which is expressed by the thermosensory AFD neurons. A: anterior, V: ventral, scale bar is 20 µm. The <i>Prom_gcy-36::cameleon</i> transgene used was <i>wzEx39</i> and the <i>Prom_gcy-18::gfp</i> transgene was <i>wzEx40</i>. (B) Expression of <i>gcy-9</i> in either the URX oxygen sensors or the AFD thermosensors rescues the behavioral defect of <i>ets-5</i> mutants. The left plot shows the mean fraction of animals ± SEM that reversed during a four second exposure to either control atmosphere (0% CO₂, 20% O₂, balance N₂) or CO₂-enriched atmosphere (10% CO₂, 20% O₂, balance N₂). The right plot shows the effect of CO₂ on reversals as measured with an avoidance index, as in Fig. 2C. Plotted are the mean avoidance indices for each of the four strains tested ± SEM. P values were calculated by one-way ANOVA. <i>N</i> = 3–5 populations of 30–50 animals. The <i>ets-</i>5 mutant strain used was FX1734. The <i>Prom</i>_{gcy<i>-36</i>}<i>::gcy-9</i> transgene used was <i>wzIs97</i> and the <i>Prom_gcy-18::gcy-9</i> transgene was <i>wzEx34</i>. (C) Expression of <i>gcy-9</i> in the URX oxygen sensors confers sensitivity to CO₂. The ratiometric calcium indicator cameleon was expressed in the URX neurons. Wild-type URX neurons (top panel) showed a small decrease in the ratio of YFP:CFP emissions in response to 10 s CO₂ pulses indicating decreases in cell calcium. URX neurons expressing <i>gcy-9</i> showed increases in cell calcium in response to CO₂ stimuli, with an average ratio change of greater than 20% (lower panel). The cameleon expression transgene used was <i>wzIs96[Prom_gcy-32::cameleon]</i> and the <i>gcy-9</i> expression transgene was <i>wzIs97[Prom_gcy-36::gcy-9].</i> (D) Expression of <i>gcy-9</i> in the AFD thermosensors confers sensitivity to CO₂. Calcium responses of wild-type AFD neurons (top panel) and AFD neurons that express <i>gcy-9</i> (bottom panel) in responses to a 10% CO₂ stimulus. The cameleon expression transgene was <i>fxIs105[Prom_gcy-8::cameleon]</i>, and the <i>Prom_gcy-18::gcy-9</i> transgene was <i>wzEx34.</i> For panels C and D, plots are mean YFP/CFP emissions ratios normalized to the pre-stimulus ratio R₀ (<i>N</i> = 16–22 animals). Red shaded areas represent SEM.</p

ETS-binding sites in the <i>gcy-9</i> promoter are required for its BAG-neuron expression.

<p>(A) Fluorescence micrographs of <i>Prom_gcy-9::gfp</i> expression in a wild-type animal and an <i>ets-5</i> mutant. <i>Prom_gcy-9::gfp</i> expression was principally in BAG neurons (top panel) and was lost in the <i>ets-5</i> mutant (bottom panel). In the lower panel, the BAGL neuron position is marked by a red circle. The scale bar in lower panel is 20 µm. A: anterior, V: ventral. The <i>ets-5</i> mutant allele was <i>tm1734</i>. The <i>Prom_gcy-9::gfp</i> transgene was <i>wzEx37</i>. (B) The ETS domain of ETS-5 is most similar to that of mammalian Pet1. The ETS domains of ETS-5 and 26 mammalian ETS-family transcription factors were identified using the SMART database. Sequences were aligned using the CLUSTALW algorithm and sequence distances were plotted as a dendrogram using the Phylip sequence analysis package. (C) The <i>gcy-9</i> promoter contains an evolutionarily conserved ETS-binding motif that is highly similar to the Pet1-binding consensus. A logo of the Pet1-binding consensus sequence <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034014#pone.0034014-Wei1" target="_blank">[24]</a> is shown aligned to sequences approximately 200 bp upstream of <i>gcy-9</i> coding sequences from <i>C. elegans</i> and three other rhabditid nematodes. (D) ETS binding motifs in the <i>gcy-9</i> promoter are required for promoter function. Shown are ventral views of adult hermaphrodites carrying a 1.9 kb fragment of the <i>gcy-9</i> promoter fused to <i>gfp</i> coding sequences, or a promoter variant that either carries an 88 bp deletion centered around the ETS binding site at −202 bp, or a variant that carries four-base substitutions in the ETS-binding sites. In each case two lines were tested for GFP expression. Red circles indicate the location of BAG neurons in the animal carrying the mutant transgene. The wild-type <i>gcy-9</i> promoter transgene used was <i>wzEx37</i> and the mutant promoter transgene was <i>wzEx38</i>.</p