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

Neuronal cell fate decisions: O2 and CO2 sensing neurons require egl-13/Sox5

We recently conducted a study that aimed to describe the differentiation mechanisms used to generate O(2) and CO(2) sensing neurons in C. elegans. We identified egl-13/Sox5 to be required for the differentiation of both O(2) and CO(2) sensing neurons. We found that egl-13 functions cell autonomously to drive O(2) and CO(2) sensing neuron fate and is therefore essential for O(2) and CO(2) sensing-induced behaviors. Through systematic dissection of the egl-13 promoter we identified upstream regulators of egl-13 and proposed a model of how differentiation of O(2) and CO(2) sensing neurons is regulated. In this commentary we discuss our findings and open questions we wish to address in future studies

Control of neuropeptide expression by parallel activity-dependent pathways in <i>Caenorhabditis elegans</i>

Monitoring of neuronal activity within circuits facilitates integrated responses and rapid changes in behavior. We have identified a system in Caenorhabditis elegans where neuropeptide expression is dependent on the ability of the BAG neurons to sense carbon dioxide. In C. elegans, CO(2) sensing is predominantly coordinated by the BAG-expressed receptor-type guanylate cyclase GCY-9. GCY-9 binding to CO(2) causes accumulation of cyclic GMP and opening of the cGMP-gated TAX-2/TAX-4 cation channels; provoking an integrated downstream cascade that enables C. elegans to avoid high CO(2). Here we show that cGMP regulation by GCY-9 and the PDE-1 phosphodiesterase controls BAG expression of a FMRFamide-related neuropeptide FLP-19 reporter (flp-19::GFP). This regulation is specific for CO(2)-sensing function of the BAG neurons, as loss of oxygen sensing function does not affect flp-19::GFP expression. We also found that expression of flp-19::GFP is controlled in parallel to GCY-9 by the activity-dependent transcription factor CREB (CRH-1) and the cAMP-dependent protein kinase (KIN-2) signaling pathway. We therefore show that two parallel pathways regulate neuropeptide gene expression in the BAG sensory neurons: the ability to sense changes in carbon dioxide and CREB transcription factor. Such regulation may be required in particular environmental conditions to enable sophisticated behavioral decisions to be performed

A Novel Role for the Zinc-Finger Transcription Factor EGL-46 in the Differentiation of Gas-Sensing Neurons in Caenorhabditis elegans

Oxygen (O(2)) and carbon dioxide (CO(2)) provoke distinct olfactory behaviors via specialized sensory neurons across metazoa. In the nematode C. elegans, the BAG sensory neurons are specialized to sense changes in both O(2) and CO(2) levels in the environment. The precise functionality of these neurons is specified by the coexpression of a membrane-bound receptor-type guanylyl cyclase GCY-9 that is required for responses to CO(2) upshifts and the soluble guanylyl cyclases GCY-31 and GCY-33 that mediate responses to downshifts in O(2). Expression of these gas-sensing molecules in the BAG neurons is partially, although not completely, controlled by ETS-5, an ETS-domain-containing transcription factor, and EGL-13, a Sox transcription factor. We report here the identification of EGL-46, a zinc-finger transcription factor, which regulates BAG gas-sensing fate in partially parallel pathways to ETS-5 and EGL-13. Thereby, three conserved transcription factors collaborate to ensure neuron type-specific identity features of the BAG gas-sensing neurons

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

The BAG neurons of <i>ets-5</i> mutants are defective in sensory transduction.

<p>(A) <i>ets-5</i> mutants are defective in a BAG-neuron-dependent CO₂ avoidance behavior. Plotted are the mean fractions 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). Strains tested were: the wild-type strain N2, the BAG-ablated strain CX11697, the <i>ets-5</i> mutant strain FX1734, which carries the <i>tm1734</i> deletion allele of <i>ets-5</i>, and a derivative of FX1734 that carries the <i>ets-5::gfp</i> transgene <i>wzIs80</i>. <i>N</i> = 3–5 populations of 30–50 animals. (B) The effect of <i>ets-5</i> mutation on CO₂ avoidance behavior is comparable to that of BAG neuron ablation. An avoidance index was calculated by subtracting the fraction of animals in a population that reversed in response to exposure to control atmosphere from the fraction that reversed in response to CO₂-enriched atmosphere. 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. (C) Wild-type BAG neurons show robust calcium responses to a CO₂ stimulus. Wild-type animals carrying a <i>Prom_gcy-9::cameleon</i> transgene, which drives expression of cameleon specifically in BAG neurons, were immobilized and exposed to a 10 s pulse of 10% CO₂. Plotted is the mean fractional ratio change in YFP/CFP emissions. The shaded area represents S.E.M. The cameleon expression transgene used was <i>wzIs82.</i> (D) The BAG neurons of <i>ets-5</i> mutants show reduced calcium responses to a CO₂ stimulus. Animals carrying a variant <i>Prom_gcy-33::cameleon</i> transgene, which drives <i>ets-5-</i>independent expression of cameleon in BAG neurons, were immobilized and exposed to a 10 s pulse of 10% CO₂. Plotted is the mean fractional ratio change in YFP/CFP emissions. The shaded area represents S.E.M. The cameleon expression transgene used was <i>wzEx56</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