The following work is my participation in the project of Audrey Bellier, on the resistance to anoxia promoted by cyclic nucleotide gated channel (cng) loss of function. One of my goals was first to observe the suspended state of the cng-2(gnv2) mutants and wild type animals under anoxia. Second I tested mutants of each of non-soluble guanylyl cyclases (gcy) from C. elegans in anoxia to assess whether a link exists between gcys and cngs. Finally I was able to assess the resistance of wild type and cng mutant homologs in Drosophila melanogaster under anoxia.
Description of cng-2(gnv2); hyl-2(gnv1)
Audrey Bellier characterized gnv2 isolated from the hyl-2(gnv1) suppressor screen.
This mutation almost completely rescued the sensitivity to anoxia (Figure 1A).
Using the same approach as that for gnv3 (i.e. combination of Whole Genome Sequencing and Rapid Single Nucleotide Polymorphism mapping), she mapped gnv2 on chromosome IV and found that a substitution mutation (D322N) affecting the cyclic nucleotide gated channel cng-2 was responsible for the suppressor effect.
Interestingly, like gnv3, gnv2 appeared to be monogenic recessive but, in contrast to gnv3, the mutation conferred resistance to anoxia when segregated from the hyl-2 loss of function mutation. In fact, cng-2(gnv2) resisted up to 72 hours in anoxia (Figure 1B).
Figure 1: Adapted from Audrey Bellier
In A, cng-2(gnv2);hyl-2(gnv1) restores a good resistance to 48 hours of anoxia. The suppressor effect is not complete since the calculated pValue between cng-2(gnv2);hyl-2(gnv1) and N2 was < 0.001. In B, segregated cng-2(gnv2) shows a significant resistance to 72 h of anoxia compared to N2, cng-2(gnv2);hyl-2(gnv1) and hyl-2(gnv1). Statistics: error bars are SEM and pValue; p<0.05; 0.01 and 0.001 (*, **, ***, respectively) were considered significant.
Intriguingly, cng-2(gnv2) corresponds to the exact same mutation that lies in the human homolog CNGA-3 in patients suffering from a hereditary cone photoreceptor disorder (Wissinger et al., 2001). Although C. elegans is capable of phototaxis (Ward et al., 2008), it does not have specialized light-sensing organs. Thus, because of the strong resistance of cng-2(gnv2) to anoxia, we hypothesized that cng-2 could be implicated in sensing other aspects of the environment.
This gene is poorly characterized but it has been recently shown that the expression of cng-2::gfp was driven exclusively in head amphid sensory neurons (AWC, ASE, ASG, ASI, ASJ and ASK), which are implicated in chemosensation, thermosensation, gas sensation and phototransduction (Wojtyniak et al., 2013).
The Cyclic Nucleotide Gated channel family
Cyclic nucleotide gated (cng) channels are known to play a role in the sensory transduction of vertebrates and invertebrates (Cho et al., 2004). In vertebrates, they are Ca2+ and Na+ channels, gated by cAMP or cGMP, which are mainly involved in vision and olfaction (Biel, 2009; Komatsu et al., 1999). In C. elegans, cng-2 belongs to this family of evolutionary conserved channels including TAX2/TAX4 and cng-3, which are known to be activated by cGMP. These channels are expressed in specific sensory neurons and are known to act as environmental sensors of thermo tolerance, CO2 and O2 (Cho et al., 2004; Gray et al., 2004; Hallem et al., 2011;
Zimmer et al., 2009). We hypothesised that cng-2 could be specifically implicated in sensing the gas environment. Experiments performed in our lab allowed us to observe that cng-2(gnv2) mutants fall into an inanimate state when placed in anoxia, on average 90 minutes later than N2. Moreover, cng-2(gnv2) ‘‘wake up’’ significantly faster during re-oxygenation. Seven hours after a period of 48 hours of anoxia only 8.5% of N2 started moving whereas 67.6% in cng-2(gnv2) were already moving.
Regulation of CNGs
It is known that CNGs such as TAX-2/TAX-4 can be regulated by soluble or non-soluble guanylate cyclase (GCY), which can be sources of cGMP that influence opening of CNGs (Ortiz et al., 2006). Interestingly, it has been previously reported that GCYs are also implicated in environmental O2 sensing (Zimmer et al., 2009).
Consequently, we initiated a screen to find out whether mutated GCYs could increase the resistance to anoxia in a similar way as cng-2(gnv2). We hoped to find
which GCY might act upstream of cng-2. We tested in anoxia all the C. elegans mutant strains, available at the CGC, for soluble and non soluble guanylate cyclase.
The genes for which no mutant strain was available were tested by RNAi screen. We did not identify any gcy mutants that would display a strong resistance to 72 hours anoxia (Figure 2).
Figure 2: Representative experiment of the gcy resistance to 72 hours of anoxia screen. All gcy mutants were tested and none of them displayed any interesting increased resistance to anoxia.
Shown here, from gcy-3 to gcy-27. N2, hyl-2(gnv1) and cng-2(gnv2) were used as controls.
This result does not exclude the possibility that these cyclases may be redundant and that downregulation of two or more of them is required to prevent CNGs from functioning properly.
Downregulation of CNGs homologs in Drosophila melanogaster allow the fly to recover faster from a period of anoxia
Research performed in plants and drosophila reinforced the idea that cng-2 could be involved in sensing the environment. Indeed, growth defects were observed in Arabidopsis thaliana with mutated cng-2 homologs (Chan et al., 2003; Finka et al., 2012). But, more interesting for us was the work of the group of David Morton, which demonstrated that downregulation of a CNG homolog in neurons expressing either the soluble guanylyl cyclase 89Da or 89Db (Gyc-89Da or Gyc-89Db) in Drosophila larvae increased their escape time from a hypoxic environment compared to wild type larvae (Vermehren-Schmaedick et al., 2010). Based on these observations we decided to setup an assay to test whether flies lacking CNGs could respond differently to anoxia from wild type flies. We knocked down CG42701, CG18210 and CG42260, described as the cng-2 homologs in the fly. We separately knocked down these three genes within Gyc-89Da or Gyc-89Db expressing neurons, which were previously reported by the Morton group to be required for a normal larval hypoxia escape response (Vermehren-Schmaedick et al., 2010). Interestingly, we observed that after undergoing a period of 1 hour of anoxia the CNG-deficient adult strains were recovering better than wild type flies (Figure 3A and 3B).
Figure 3: cng RNAi CG42701, CG18210 and CG42260 are induced after crossing with either gyc-89Da on chromosome II (gyc-89Da_II) (A) or gyc-89Db on chromosome II (89Db_II) (B). The wt strain was crossed separately with drivers as a control. In both cases the RNAi-induced strains are undergoing 1 h of anoxia and are observed for their ability to recover, which is represented by the number of flies able to climb above a threshold of 3 cm from the bottom of the tube. The RNAi lines show a better recovery compared to the wt control, especially with the 89Da driver.
Although I initiated these experiments, Minkyoung Lee, a Master in the lab, performed additional experiments to demonstrate that the response to anoxia in adult D. melanogaster was specifically linked to sensory neurons expressing guanylyl cyclases. In fact, downregulation of CNGs within cells expressing both 89Da and 89Db (with the driver 10XUASGFP;DaDbsph) induced a better recovery after anoxia (Figure 4A), while downregulation of CNGs with motoneuron-related driver (OK-371) did not show any effect in the recovery compared to the wild type (Figure 4B).
Figure 4: Adapted from Minkyoung Lee
cng RNAi CG42701, CG18210 and CG42260 are induced by being crossed with either 10XUASGFP;DaDbsph (A) or OK-371 (B). The wt strain was crossed separately with drivers as a control. In A, the RNAi were expressed only in neuron expressing Gyc-89Da and Gyc-89Db and induced a better recovery after 1 hour of anoxia compared to the wild type. In B, the RNAi were expressed with motoneuron-related driver OK-371 and did not show better recovery.
Altogether, these results suggest that, at least part of the response to anoxia is conserved between C. elegans and D. melanogaster.
Conclusion
These observations have revealed a common important role of cng-2 in the recovery after a period of anoxia in C. elegans and in Drosophila. Interestingly, phototransduction in the fly requires light-sensitive TRP channels (Montell, 1999), while vertebrates use light-sensitive CNG channels (Fu and Yau, 2007). This shows the possibility of different roles of CNGs in other transduction pathways throughout evolution.
Material and Methods of Appendix 3
Anoxia experiment with C. elegans
Anoxia experiments were performed as described in (Menuz et al., 2009). All tested animals were in between 25-30 synchronised 72L1 per strain. Survivors were scored after recovering for a period of 24 h in normoxic environment. Observations were done either with a binocular (Leica MZ6) or with an inverted fluorescent microspcope (Zeiss Axiovert).
RNAi gcy screen on C. elegans
RNAi experiments were performed in solid NGM plates enriched with carbenicilin (25µg/ml final concentration) and IPTG (1mM final concentration). Worms were fed with HT115 strains containing either the empty vector L4440 as a control or with L4440 containing clones from C. elegans RNAi library obtained at Source Bioscience LifeScience and daf-2 clone made by (Dillin et al., 2002).
Experiments were performed at 20°C following the Ahringer lab RNAi Feeding Protocol (Version 11.04.01) based on (Kamath et al., 2001).
RNAi experiment with D. melanogaster
Downregulation of cng-like homologues CG42701, CG18210 and CG42260 was performed as described in (Vermehren-Schmaedick et al., 2010), with the balancers previously removed from the driver strains, this was to avoid any influence of the genomic background on the experiment. The drivers used were: 89Da on II, 89Db on II, OK-371 and 10XUASGFP;DaDbsph.
Anoxia experiment with D. melanogaster
Anoxic environment was generated inside a Modular incubator chamber (Billups-Rothenberg) by flushing out oxygen with 5.0 nitrogen gas at a rate of 20l/min, at a pressure of 1bar. Oxygen is removed in less than two minutes and the nitrogen flush is maintained throughout the experiment.
Tests were performed with pseudo synchronized young adult flies (50% females + 50% males) which were placed into tubes (30 per tubes) containing fly food capped with a grid to optimize air circulation. Anoxia was performed for 1 hour and fly tubes were removed from the incubator and flies were observed for their recovery abilities for 50-60 minutes.
Observation of fly recovery after 1 hour of anoxia
Tubes were vertically disposed and the recovery was measured looking at the ability of flies to spontaneously climb up the wall of the tube. Every 5 minutes, the number of flies able to climb over 3 cm from the bottom of the tube was scored.