Article Figures & Data
Figures
- Figure 1.
A subset of stomatin domain genes are expressed in C. elegans olfactory neurons. A, Domain organization of stomatin domain genes. Colored in shades of gray are the central conserved stomatin domain and the hydrophobic domain that mediates association with the membrane. Flanking these are the divergent N and C termini. B, Gene tree for family of stomatin domain gene homologues, TF105750 (stomatin-like 2) from TreeFam (Ruan et al., 2008). Some organism silhouettes created with BioRender. C, D, RNA Seq data from CenGEN consortium sorted neuron populations, mapped to the worm genome with STAR and gene reads counted by HTSeq. C, Olfactory neurons AWA and AWB. D, Motor neurons DD and VD. See also Extended Data Figure 1-1A for comparison with olfactory neuron marker gene FPKM. E, Cluster of three Stoml3/Stomatin-like genes in the C. elegans genome on chromosome X. F, Two immediately adjacent Stoml3/Stomatin-like genes in the Drosophila genome on chromosome X. See also Extended Data Figure 1-1.
- Figure 2.
Specific stomatin domain genes required for olfaction in C. elegans. A, Schematic of the chemotaxis assay. Worms are placed in the center of an unseeded worm plate, given a choice between point sources of odor (represented by +) and unscented diluent (represented by 0), and given 1 h to chemotax. B, C, Chemotaxis assays for the attractive odor pyrazine (1:1000; B) or the repulsive odor octanol (1:1000; C). The chemotaxis index = ((worms in + quadrant)(worms in 0 quadrant))/(worms in all quadrants), with a maximum value of 1 (attractive) and minimum value of −1 (repulsive). D, Schematic of the smell on a stick assay for repulsive odors. E, Smell on a stick assay for octanol. F, AWA neurons labeled by translational ODR-10::GFP fusion. White arrowheads denote sensory cilia (where ODR-10 localizes). Gray arrowheads denote cell bodies. The cell body in the mec-2 image is slightly out of the plane of focus. Scale bar represents 50 μm. See also Extended Data Figure 2-1C,D. G, GCaMP5 imaging of AWC(on) neurons using a microfluidic “olfactory chip.” Isoamyl alcohol (1:1000) administration represented by the gray box. Error bars represent SEM N = 12 young adult worms (5 wild-type, 7 mec-2 mutants). F, Schematic of mec-2 alternative splicing and (G) subsequent protein isoforms. H, I, mec-2 alternative isoforms at the level of pre-mRNA splicing (H) and protein isoforms (I). The isoforms affect the divergent C terminus of mec-2. J, CenGEN RNA Seq data reveals mec-2 is found primarily as the mec-2B isoform in AWA and AWB olfactory neurons. K, Smell on a stick assay, 1:100,000 octanol, statistical test (ANOVA) performed against mec-2() mutants. Re-expression of any mec-2 isoform in AWB neuron partially rescues response. Asterisks indicate p < 0.05, one-way ANOVA. See also Extended Data Figure 2-1.
- Figure 3.
Stomatin domain genes expressed in mouse neurons, and Stoml3 knock-out mice. A–D, Cell-specific sequencing of mouse olfactory neurons (A, B) and motor neurons (C, D). Omp is used as a positive marker for olfactory neurons, and Chat as a positive marker for cholinergic motor neurons. E, Stoml3 gene in mouse, indicating the region we deleted in orange, and the primer sets used to detect both the wild-type and mutant genomic DNA (black arrows). F, Sanger sequencing confirming the ∼5.4-kb deletion. G, Genotyping gels for identifying homozygous Stoml3 mutants and their wild-type littermates.
- Figure 4.
Olfactory assays for wild-type and Stoml3 knock-out mice. A, B, Innate olfactory attraction tests. Mean time sniffing a scented block in wild-type mice (n = 16) and Stoml3 knock-out mice (n = 11) during the 3-min test period. Water was used as a control scent. Peanut butter and mouse urine were used as attractive scents. Both wild-type and Stoml3 knock-out mice showed attraction response to peanut butter and mouse urine. Asterisk indicates p < 0.05. C, Buried cereal test. Mean time wild-type (n = 14) and Stoml3 knock-out mice (n = 10) took to find the pellet. Surface pellet test is a positive control, confirming that the cereal pellet is attractive to the mice. ns represents no significant difference between wild types and Stoml3 knock-outs using Mann–Whitney U test. D, Habituation/dishabituation test. Mean time sniffing a scented cartridge in wild-type mice (n = 16) and Stoml3 knock-out mice (n = 12) during 30-s test period across seven trials. Almond extract was used on trials 1–6, and banana extract was introduced as a novel scent on trial 7. Significant difference of sniffing time in wild types is observed when novel scent is introduced on trial 7 (**p < 0.01), while no significant difference is observed between trial 6 and trial 7 in Stoml3 knock-out mice. E, Block test. Mean time sniffing home cage blocks A–C and novel block E in wild-type mice (n = 16) and Stoml3 knock-out mice (n = 12). Wild types spend more than 2-fold time exploring novel block (E) than Stoml3 knock-out mice (**p < 0.01, Mann–Whitney U test, error bars represent SEM). See also Extended Data Figure 4-1.
Extended Data
Extended Data Figure 1-1
Expression and location of Stomatin domain gene homologues. A, FPKMs for osm-6 as a positive control for a gene highly expressed in olfactory neurons (it serves as a sensory neuron marker gene) and lowly expressed in other neurons, including motor neurons. B, Additional cluster of Drosophila Podocin homologues on chromosome 3. Download Figure 1-1, TIF file.
Extended Data Figure 2-1
Stomatin domain genes required for behavior in C. elegans. A, Fraction of worms that fail to leave the 1-cm diameter origin circle after 1 h. Note that the majority of sto-4 and unc-1 mutants fail to locomote away from origin, whereas both wild-type and chemotaxis defective mutants do not remain in origin. B, Maximal ΔF/F GCaMP signals during odor presentation of isoamyl alcohol (1:1000) as displayed in Figure 2G. Unpaired two-tail t test, p < 0.05. C, D, AWB and AWC neuronal cell bodies visualized by transgene oyIs44 (ord-1p::RFP), revealing no obvious morphological differences in cell body position, axons, or dendrites between wild-type (C) and mec-2 mutant worms (D). Download Figure 2-1, TIF file.
Extended Data Figure 4-1
Stoml3 behavior and mec-2 expression. A, Latency to uncover pellet in cereal test measured on a daily basis, showing no significant differences between dynamics of wild-type and Stoml3 KO mice. B, In trial 6 of the block test, where no novel odor is present, time spent exploring the blocks is minimal. C, Single-cell sequencing data from CenGEN consortium data on mec-2 expression in representative neuron populations. Size of dot represents proportion of single cells in which mec-2 was detected, and heatmap represents scaled TPM of the mec-2 gene. A few sensory cell types are highlighted. Download Figure 4-1, TIF file.
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