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.

BackgroundInhibitors of nicotinamide phosphoribosyltransferase (NAMPT) are promising cancer drugs currently in clinical trials in oncology, including APO866, CHS-828 and the CHS-828 prodrug EB1627/GMX1777, but cancer cell resistance to these drugs has not been studied in detail.MethodsHere, we introduce an analogue of CHS-828 called TP201565 with increased potency in cellular assays. Further, we describe and characterize a panel of cell lines with acquired stable resistance towards several NAMPT inhibitors of 18 to 20,000 fold compared to their parental cell lines.ResultsWe find that 4 out of 5 of the resistant sublines display mutations of NAMPT located in the vicinity of the active site or in the dimer interface of NAMPT. Furthermore, we show that these mutations are responsible for the resistance observed. All the resistant cell lines formed xenograft tumours in vivo. Also, we confirm CHS-828 and TP201565 as competitive inhibitors of NAMPT through docking studies and by NAMPT precipitation from cellular lysate by an analogue of TP201565 linked to sepharose. The NAMPT precipitation could be inhibited by addition of APO866.ConclusionWe found that CHS-828 and TP201565 are competitive inhibitors of NAMPT and that acquired resistance towards NAMPT inhibitors can be expected primarily to be caused by mutations in NAMPT.

Animals harbor specialized neuronal systems that are used for sensing and coordinating responses to changes in oxygen (O2) and carbon dioxide (CO2). In Caenorhabditis elegans, 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. egl-13 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 egl-13 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, egl-13, is required and sufficient to specify the distinct fates of O2- and CO2-sensing neurons in C. elegans. These findings extend our understanding of mechanisms of neuronal diversification and the regulation of molecular factors that may be conserved in higher organisms.

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.T HE acquisition of specific neuronal fates is controlled by regulatory transcription factors that activate the expression of neuron type-specific terminal differentiation genes (Hobert 2008(Hobert , 2011. Specific individual transcription factors may act to control the expression of the entire terminal gene battery of a specific neuron, or alternatively, multiple transcription factors may act in parallel to control distinct aspects of terminal fates . The use of combinatorial transcription factors to control aspects of terminal fates may provide flexibility to polymodal neurons that need to rapidly respond to changes in a specific environmental cue. We study the BAG neurons in Caenorhabditis elegans that have a dual function where they are activated by separate mechanisms when carbon dioxide (CO 2 ) levels increase or when oxygen (O 2 ) levels decrease (Zimmer et al. 2009). The membrane-bound receptor-type guanylyl cyclase GCY-9 is expressed specifically in the BAG neurons to mediate CO 2 avoidance behavior . Whereas the soluble guanylyl cyclases GCY-31 and GCY-33 are expressed in the BAG neurons to mediate behavioral responses when O 2 levels decrease (Zimmer et al. 2009). Previous studies have shown that the ETS domain-containing transcription factor ETS-5 is critical for CO 2 -sensing neuron fate through direct regulation of GCY-9 expression in the BAG neurons (Guillermin et al. 2011; Brandt et al. 2012;Gramstrup Petersen et al. 2013). In addition, the Sox transcription factor EGL-13 is partially required for GCY-9 expression in the BAG neurons (Gramstrup Petersen et al. 2013). Loss of either of these transcription factors causes defects in CO 2 avoidance behavior (Guillermin et al. 2011; Brandt et al. 2012;Gramstrup Petersen et al. 2013). The O 2 -sensing neuron fate of the BAG neurons, as assessed by expression of gcy-31 and gcy-33, is also defective in ets-5 and egl-13 mutant animals (Gramstrup Petersen et al. 2013). However, the expression of these and other B...

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, CO2 sensing is predominantly coordinated by the BAG-expressed receptor-type guanylate cyclase GCY-9. GCY-9 binding to CO2 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 CO2. 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 CO2-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.