Figure S2. Notch Receptors Act in Neurons to Regulate Octanol Response, Related to Figure 2 Time to respond to octanol is reported as mean time to initiate backward locomotion (s) +/- SEM. Sample size for each genotype or treatment is given on each bar. (A)
glp-1 is likely expressed in neurons. A GFP reporter was constructed containing 2.5 kb of putative regulatory sequence upstream and downstream of the
glp-1 translation start site (
glp-1p::gfp; see Supplemental methods for details). GFP expression was observed near the excretory pore, anus, in vulval precursor cells, intestinal cells, and in a few head neurons that could not be identified due to low expression levels. Previous studies indicated that
glp-1 activation increases
glp-1 transcription [11,12,13,14]. As the
glp-1 reporter construct described above contains numerous binding sites for LAG-1, a C. elegans homolog of the CSL/Su(H) transcription factor homolog that is critical for Notch transcriptional activation of target genes, we reasoned that over-expressing
osm-11 might lead to increased
glp-1p::gfp expression. Within hours heat shock induction of OSM-11 expression, GFP expression was clearly observed in many neurons of hsp::
osm-11;
glp-1p::gfp transgenic animals. These neurons included ASG, ASH, ASI, ADL, ASJ, ADF, and ASE sensory neurons, but not the RIG interneurons that express
lin-12. The ASH, ADL, and AWB sensory neurons detect octanol semi-redundantly [4,15] suggesting that
glp-1 might act to modulate octanol response in these sensory neurons. Shown here is
glp-1p::gfp expression in head neurons (arrow indicates ASHR ). Scale bar represents 10 m. (B)
glp-1(RNAi) in subsets of neurons had modest impact on octanol response (
osm-10p::
glp-1(RNAi) in ASH, ASI, PHA, PHB sensory neurons;
gpa-11p::
glp-1(RNAi) in ASH and ADL only). *p < 10-13 vs. WT, #p < 0.025 vs.
lin-12(null), @p < 0.004 vs.
lin-12(null);
gpa-11p::gfp. Germline morphology, fertility and vulval morphology were not visibly altered by neuronal
glp-1(RNAi) constructs, suggesting that no dsRNA spreading occurred (data not shown). (C)
osm-11 acts via Notch receptors. The two Notch gain of function alleles:
lin-12(
n137n460), a non-constitutive, cold-sensitive gain of function (csgf) allele, and
glp-1(
ar202), a temperature-sensitive, gain of function (tsgf) allele were used to determine if
osm-11 acts via Notch receptors. The gain of function alleles had little or no effect on octanol response in otherwise normal animals regardless of temperature. Conditional gain-of-function alleles
lin-12(csgf) and
glp-1(tsgf) (reared at restrictive temperature of 15oC and 25C, respectively) partially suppressed the octanol response defects of
osm-11(null) animals. p < 0.01 vs. WT. p < 0.05 vs.
osm-11(null) reared at 15C; &p < 0.05 vs.
osm-11(null) reared at 25C. (D)
lag-2p::gfp is expressed in many head neurons of adult C. elegans including the AVA, one of the command interneurons (arrow) that regulates locomotion and helps initiate reversals in response to aversive stimuli. Scale bar represents 10 m. (E)
egl-4 and
lst-1, are two candidate genes that act downstream of Notch and express in neurons.
egl-4 encodes a cGMP-dependent protein kinase (PKG)[16] that is expressed in many head neurons and has been previously implicated in several C. elegans behaviors. The gain-of-function
egl-4(
ad450) allele partially suppressed the octanol response defect of
osm-11(null) animals suggesting that
egl-4 likely acts downstream of Notch signaling pathway in the regulation of octanol response. As no obvious changes in
egl-4p::gfp expression were observed in Notch receptor mutant backgrounds (data not shown), either the reporter gene was not sensitive enough to detect modest changes in gene expression, the regulation occurs at a post-transcriptional level, or
egl-4 is not directly downstream. Next, the role of
lst-1 was tested. If
lst-1 [17] is a direct target of Notch receptors in neurons, then increasing Notch receptor signaling should have no effect on octanol response in animals lacking
lst-1. However, the gain-of-function
lin-12(
n137n460csgf) allele suppressed the octanol response defects of
lst-1(null) animals, as did over-expression of
osm-11. Thus,
lst-1 likely works in parallel with other genes downstream of Notch signaling in octanol response. *p < 10-3 vs. WT; p < 0.001 vs.
osm-11(null); #p < 0.01 vs.
lst-1(null).