, 2006). These appear to be O2 regulated ( Gray et al., 2004 and Boon and Marletta, 2005) because both are required for BAG O2 responses ( Zimmer et al., 2009). To examine if GCY-31, GCY-33, or both are required in CO2 sensory transduction, we imaged BAG responses to 3% CO2 in gcy-31; gcy-33 double-deletion mutants. Loss of gcy-31 and gcy-33 reduced the CO2-evoked BAG Ca2+ response ( Figures 5B and 5C). This suggests that GCY-31 and/or GCY-33 forms part of the CO2 sensory system in BAG, although other molecules are likely to be involved. We next imaged AFD responses in tax-2(null) and tax-2(p694) animals. NVP-BKM120 supplier Expression from the gcy-8 promoter is markedly reduced in tax-2 and tax-4 mutants

( Satterlee et al., 2004), and YC3.60 expression was correspondingly low in AFD in tax-2(ot25null) animals. In contrast, expression in tax-2(p694) animals was similar to wild-type (data not shown). Both tax-2 mutations significantly reduced the AFD CO2 response, but neither completely abolished it ( Figures 5D–5F). The AFD ON-minimum appeared to be absent in both tax-2 mutants, whereas the AFD ON-maximum was absent in tax-2(null) animals but enhanced in tax-2(p694) animals ( Figures 5D–5F). Our data suggest that all three components of the AFD

CO2 response involve TAX-2 mediated cGMP pathways but that other pathways also contribute. To further investigate molecular mechanisms mTOR inhibitor of CO2 sensing, we asked whether C. elegans CO2 sensors express carbonic anhydrases, hallmarks of CO2-responsive neurons in other animals ( Hu et al., 2007, Wang et al., 2002, Ridderstrale and Hanson, 1985 and Coates et al.,

1998). Database searches indicate that the C. elegans genome encodes eight predicted carbonic anhydrases. Six, cah-1 to cah-6, belong to the alpha family, and two, bca-1 and bca-2, to the beta family. Because many members of the beta family are mitochondrial ( Syrjänen et al., 2010 and Fasseas et al., 2010), we focused our studies on the alpha family. We fused upstream promoter regions of each gene to gfp and examined the resulting expression patterns. We found that cah-1, 2, 3, and 6 show strong neuronal expression in adults ( Figure S3A). cah-4 was primarily expressed in the hypodermis (excluding the seam cells) and in the excretory Chlormezanone cell, consistent with a kidney-like function for this cell. cah-3 and cah-5 show expression in intestinal cells, with cah-3 expression being especially strong. Using a pBAG::mCherry marker, we showed that cah-2, but not apparently any of the other five cah genes, was expressed in BAG ( Figure S3B). cah-2 was also expressed in a set of four quadrant head neurons, other unidentified head neurons, the canal neurons CANL/R, whose processes run parallel to the tracts of the excretory cell, and a pair of tail neurons ( Figure S5). Previous data suggest that cah-2 is also expressed in AFD ( Colosimo et al., 2004).