Figure 3. if-1⁺/cali⁺ cells express neurotransmitter reuptake and metabolism genes.

(A–N) Schematic indicates region of focus. (A–H) Double FISH of if-1/cali (magenta) and neural markers (A) pc2, (B) chat, (C) syn, (D) syt1-1, (E) syt1-2, (F) syngr, (G) SNAP25, and (H) unc-13 (green). No co-expression was observed between neural markers and if-1 and cali. (I–N) Double FISH of if-1/cali (magenta) with orthologs of vertebrate astrocyte markers (I) gs, (J) eaat2-1, (K) eaat2-2, (L) gat, (M) glut, and (N) trpm (green). Lower panels show high magnification images of, from left to right, if-1/cali (magenta), astrocyte marker ortholog (green), DAPI (blue), and merged channels from a representative double-positive cell. (O–W) Schematic indicates region of focus. The images show one hemisphere of the cephalic ganglia and the lateral parenchymal space. White dotted line delineates the edge of animal. Yellow dotted line delineates borders of the neuropil. (O–S) Double FISH of gs (magenta) with (O) gat, (P) eaat2-1, (Q) eaat2-2, (R) glut, and (S) trpm (green). 98.7 ± 1.4% of glut⁺ cells in the neuropil and 99.7 ± 0.7% of glut⁺ cells outside the neuropil expressed gs. 96.8 ± 4.6% of trpm⁺ cells in the neuropil and 93.8 ± 4.5% of trpm⁺ cells outside the neuropil expressed gs. Arrowheads denote double-positive cells outside the neuropil. Lower panels show high magnification images of, from left to right, gs (magenta), astrocyte marker ortholog (green), DAPI (blue), and merged channels from a representative double-positive cell. (T–U) Double FISH of ptc (green) with (T) gs and (U) glut (magenta). 99.3 ± 0.7% of glut⁺ cells in the neuropil and 90.4 ± 4.5% of glut⁺ cells outside the neuropil expressed ptc. Arrowheads denote double-positive cells. Lower panels show high magnification images of, from left to right, gs or glut (magenta), ptc (green), DAPI (blue), and merged channels from a representative double-positive cell. (V–W) Double FISH of pc2 (green) with (V) gs and (W) eaat2-1 (magenta). No double-positive cells were observed in both cases. Anterior up, ventral side shown for all. Maximum intensity projections shown for I–N. Scale bars: 100 um for overviews, 10 um for insets for A–N; 50 um for overviews, 10 um for insets for O–W.

DOI: http://dx.doi.org/10.7554/eLife.16996.014

Figure 3—figure supplement 1. if-1 and cali expression does not overlap with neuronal marker expression.

(A–B) Double FISH for if-1/cali and (A) pc2 or (B) syn. Each row shows high magnification images of, from left to right, if-1/cali (magenta), pc2 or syn (green), DAPI (blue), and merged channels from a representative cell cluster. (C) Double FISH for if-1/cali and other described neuronal markers in wild-type untreated animals. See Figure 1—source data 1 for more information of neural markers used in co-expression studies. Anterior up, ventral side shown for C. Scale bars: 10 um for A, B; 100 um for C.

DOI: http://dx.doi.org/10.7554/eLife.16996.020

Figure 3—figure supplement 2. Expression patterns of markers for if-1⁺/cali⁺ cells.

(A–F) WISH for (A) gs, (B) eaat2-1, (C) eaat2-2, (D) gat, (E) glut, and (F) trpm. Black arrowheads indicate light staining. (G) gs(RNAi) animals display reduced gs expression in the CNS compared to control animals (n = 4/4). (H) gat(RNAi) animals display slightly reduced gat expression in the CNS compared to control animals (n = 4/5). (I) eaat2-1(RNAi) animals display reduced eaat2-1 expression in the CNS compared to control animals (n = 9/9). (J) eaat2-2(RNAi) animals display reduced eaat2-2 expression in the CNS compared to control animals (n = 6/6). Anterior up, ventral side shown. Scale bars: 500 um.

DOI: http://dx.doi.org/10.7554/eLife.16996.021

Figure 3—figure supplement 3. Maximum likelihood cladogram for excitatory amino acid transporters.

S. mediterranea EAAT2-1 and EAAT2-2 fall within the excitatory amino acid transporter 2 clade.

Neutral amino acid transporter proteins SLC1A5 and SLC1A6 were used as an outgroup to root the tree. Topology of vertebrate excitatory amino acid transporters roughly recapitulates previous results (Gesemann et al., 2010). Bootstrap support values listed at branch junctions. Accession numbers of protein sequences used in the analysis listed in Figure 3—source data 2.

DOI: http://dx.doi.org/10.7554/eLife.16996.022

Figure 3—figure supplement 4. Maximum likelihood cladogram for GABA transporters.

S. mediterranea GAT is placed with other Protostome GABA transporters, which have diverged from the vertebrate branch that includes GAT-2, GAT-3, CT1, and TAUT. Dopamine transporter DAT1 was used as an outgroup to root the tree. Topology roughly recapitulates previous results (Kinjo et al., 2013). Bootstrap support values listed at branch junctions. Accession numbers of protein sequences used in the analysis listed in Figure 3—source data 3.

DOI: http://dx.doi.org/10.7554/eLife.16996.023

Figure 3—figure supplement 5. Maximum likelihood cladogram for glucose transporters.

S. mediterranea GLUT clusters with other Lophotrochozoan glucose transporters, which diverged prior to the vertebrate glucose transporter radiation. Fructose transporter GLUT5 was used as an outgroup to root the tree. Bootstrap support values listed at branch junctions. Accession numbers of protein sequences used in the analysis listed in Figure 3—source data 4.

DOI: http://dx.doi.org/10.7554/eLife.16996.024

Figure 3—figure supplement 6. Maximum likelihood cladogram for transient receptor potential channels.

S. mediterranea TRPM falls outside the cluster of other TRPM proteins but apart from the nearest subfamily, TRPC. Maximum likelihood tree of transient receptor potential channels constructed by PhyML with 1000 bootstrap replicates. Mucolipins were used as an outgroup to root the tree. Topology roughly recapitulates previous results (Matsuura et al., 2009). Bootstrap support values listed at branch junctions. Accession numbers of protein sequences used in the analysis listed in Figure 3—source data 5.

DOI: http://dx.doi.org/10.7554/eLife.16996.025