A cellular and regulatory map of the cholinergic nervous system of C. elegans
- Columbia University, United States
- Howard Hughes Medical Institute, Columbia University, United States
- Weizmann Institute of Science, Israel
- Albert Einstein College of Medicine, United States
- MRC Laboratory of Molecular Biology, United Kingdom
- Texas A&M University, United States
- Howard Hughes Medical Institute, Texas A&M University, United States
Figures
Figure 1 with 3 supplements
Expression of cholinergic pathway genes in the adult C. elegans hermaphrodite.
(A) Cholinergic pathway genes. Ch = choline; ACh = acetylcholine; ChAT = choline acetyltransferase; VAChT = vesicular ACh transporter, AChE = ACh esterase, ChT = choline transporter. (B) Fosmid reporters used in this study. The unc-17 fosmid reporter was kindly provided by the TransgeneOme project (Sarov et al., 2012). It was previously reported that the expression of unc-17/VAChT and cha-1/ChAT overlap completely (Mathews et al., 2015). (C) unc-17 and cho-1 fosmid reporter expression in an L4 hermaphrodite. The fluorescent reporter inserted into the cho-1 locus is targeted to the nucleus (see Materials and methods), while the fluorescent reporter inserted into the unc-17 locus is fused directly to the unc-17 gene (resulting in cytoplasmic localization). (D, E) unc-17 and cho-1 fosmid reporter expression in head (D), retrovesicular ganglion and tail ganglia (E). In (E) bottom panels, neurons are labeled with a green pan-neuronal marker, ric-19. Transgenes: otIs576 = unc-17 fosmid reporter; otIs544 = cho-1 fosmid reporter, otIs380 = ric-19 reporter (Stefanakis et al., 2015). (F) Immunofluorescent staining for endogenous UNC-17 protein of unc-104(e1265) animals that express the cho-1 fosmid reporter transgene otIs544. (G) Co-labeling cholinergic (cho-1/ChT-positive) and glutamatergic (eat-4/VGLUT-positive) neurons illustrate no overlap in neurotransmitter ACh and Glu expression, and co-labeling with pan-neuronal marker rab-3 illustrates that most neurons now have a neurotransmitter assignment. Transgenes: otIs544 = cho-1 fosmid reporter, otIs388 = eat-4 fosmid reporter (Serrano-Saiz et al., 2013), otIs355 = rab-3 reporter. (H) ace/AChE genes are expressed in a subset of cholinergic neurons and in non-cholinergic neurons. ace-1 fosmid reporter expression in head neurons (left panel). ace-2 fosmid reporter expression in head neurons together with cho-1 fosmid reporter (middle panel). ace-3/4 reporter expression together with cho-1 fosmid reporter in head neurons (right panel). Transgenes: otEx4435 = ace-1 fosmid reporter; otEx4431 = ace-2 fosmid reporter; fpIs1 = ace-3/4 transcriptional reporter.
Figure 1—figure supplement 1
Neuronal cell identification.
Neuronal identity was confirmed by crossing cho-1 fosmid reporter and/or unc-17 fosmid reporter with specific markers. (A) ADF and RIH were labeled by cho-1 (otIs354) and cat-1 (otIs625). (B) ASJ and AWB were labeled by cho-1 (otIs354) and DiI staining. (C) AVA, AVE and AVD were labeled by cho-1 (otIs544) and nmr-1 (akIs3). (D) AVA, AVE and AVD were also labeled by cho-1 (otIs544) and glr-1 (hdIs30). (E) AVB was labeled by cho-1 (otIs544) and acr-15 (wdEx290). (F) AVB was also labeled by cho-1 (otIs544) and sra-11 (otIs123). (G) AVB was not labeled by glr-1(hdIs30) as had been previously published (Brockie et al., 2001). (H) AWA was labeled by odr-10 (kyIs37) but did not show cho-1 (otIs544) expression. (I) AWA was labeled by gpa-4 (otEx6381) but did not show unc-17 (otIs576) expression. (J) AVG and RIF were labeled by cho-1 (otIs544) and odr-2 (otEx4452). (K) DVA was labeled by ser-2 (otIs358) and cho-1 (otIs544). (L) PDA was labeled by cho-1 (otIs544) and ace-3/4 (fpIs1). (M) ALN and PLN were labeled by cho-1 (otIs544) and lad-2 (otIs439). (N) SMB and SMD were labeled by cho-1 (otIs544) and lad-2 (otIs439). (O) SMD and RIV were labeled by cho-1 (otIs544) and lad-2 (otIs439). (P) SIA and SIB were labeled by cho-1 (otIs544) and ceh-24 (ccIs4595). (Q) URX, RIR and RIH were labeled by cho-1 (otIs544) and unc-86 (otIs337). (R) VC4 and VC5 were labeled by cat-1 (otIs221) but not by cho-1 (otIs544).
Figure 1—figure supplement 2
Neurotransmitter identity of pharyngeal neurons.
(A) The different panels show the expression of unc-17 (otIs576) and cho-1 (otIs544) fosmids in the pharyngeal neurons in the anterior and posterior bulbs. Only M4 and M5 express both fosmids. Schematic for the cholinergic pharyngeal neurons is shown. (B) Expression of eat-4 (otIs518) and cho-1 (otIs344) fosmids in the pharyngeal neuron M5.
Figure 1—figure supplement 3
Expression of unc-17 and cho-1 fosmid reporters in the male tail.
(A) The top panel shows the male pre-anal ganglion on a ventral view and the bottom panel shows the pre-anal ganglion and tail neurons in a lateral view. (B) Male tail ventral view where PVS, PVU and the male-specific neurons PVZ and HOB were labeled by cho-1 fosmid and ida-1::gfp reporter. Transgenes: otIs576 = unc-17 fosmid reporter; otIs544 = cho-1 fosmid reporter; inIs179 = ida-1 reporter.
Figure 2
Distribution of neurotransmitters throughout the nervous system of the hermaphrodite.
(A) Pie chart with numbers/distributions of cholinergic (this study), glutamatergic (Serrano-Saiz et al., 2013), GABAergic (McIntire et al., 1993) and aminergic (Chase and Koelle, 2007) neurons (including pharyngeal neurons). Inset: Pie charts of extrapharyngeal sensory, motor- and interneurons. Neurons that contain a classic fast transmitter plus an aminergic transmitter (e.g. RIH) are counted in the fast transmitter category. Classification of C. elegans neurons into sensory, inter- and motor neurons is complicated by the fact that a subset of sensory neurons are also motor neurons, i.e. synapse directly onto muscle (we count those neurons here only as sensory neurons). Conversely, a large number of motor neurons also extensively synapse onto other motor neurons or interneurons and hence classify as 'interneuron' as well; these neurons are shown exclusively in the motor neuron category. A number of neurons that were originally assigned as 'interneurons' by John White and colleagues are now considered motor neurons (because of the more recent identification of NMJs; e.g. SIA, SIB, SAB neurons), or are considered sensory neurons (because of their position in connectivity diagrams or expression of molecular markers; e.g. URA, URB, URXY, URY). See Table 2 for a complete list of neurons and their neurotransmitter assignment. Lastly, we note that unpublished results from our lab demonstrate that at least two additional interneurons, not shown here, utilize GABA (M. Gendrel and O.H., unpubl. data). (B) Distance of sensory neurons to motor output (processing depth) of cholinergic and glutamatergic sensory neurons. (C) Location of neurons with different neurotransmitter identities in the head ganglia. (D) Neurotransmitter identity does not track with lineage history. Neurotransmitter identity is superimposed on the embryonic lineage diagram (Sulston et al., 1983), with each color line indicating one neuron type with a defined identity. White lines indicate no known neurotransmitter identity, gray lines indicate non-neuronal cells. Lines with two colors illustrate co-transmitter identities.
Figure 3
Neurotransmitter distribution in nervous system-wide circuit diagrams.
(A, B) Circuit diagrams, taken from White et al. (1986), with neurotransmitter identities added in colors, as indicated. Panel A shows what White et al. called the “Circuitry associated with motoneurons in the nerve ring” and panel B shows the “Circuitry associated with the motoneurons of the ventral cord”. (C) A visualization of the C. elegans connectome that reflects signal flow through the network as well as the closeness of neurons in the network, as previously proposed and described (Varshney et al., 2011). Coordinates from the diagram were kindly provided by Lav Varshney. The vertical axis represents the signal flow depth of the network, i.e. the number of synapses from sensory to motor neurons. The horizontal axis represents connectivity closeness. We superimpose here neurotransmitter identity onto this network diagram, illustrating some network cluster enriched for ACh usage (shaded gray). (D) A graphic representation that focuses on processing depth, illustrating whether a neurotransmitter is used more frequently in upper (sensory) or lower (motor) layers of the network. (E) Network motifs enriched in the C. elegans connectome and their neurotransmitter usage. Colors indicate if the neurons in this position are enriched for the usage of Glu or ACh.
Figure 4
Expression pattern of ACh-gated chloride channels.
Expression pattern of acc fosmid reporters in L4 stage animals are shown. Transgenes: otEx6374 = acc-1 fosmid reporter; otEx6375 = acc-2 fosmid reporter; otEx6376 = acc-4 fosmid reporter; otIs545 = cho-1 fosmid reporter; otIs518 = eat-4 fosmid reporter. Besides the neurons shown here, acc-1 and acc-2 are expressed in a small number of additional neurons (not shown).
Figure 5
Sexual and temporal dynamics of cholinergic identity.
(A) Male-specific CEM neurons are cholinergic, but turn on cho-1 (otIs544) and unc-17 (otIs576) only in late L4. In the top panels CEM neurons are labeled by the pkd-2 reporter (bxIs14). See Figure 1—figure supplement 3 and Table 5 for a list of all male-specific cholinergic neurons. (B) Hermaphrodite-specific HSN neurons turn on the cholinergic marker unc-17 and pan-neuronal rab-3 also in late L4. HSN neurons are labeled by a nuclear localized unc-86 fosmid reporter (otIs337). At L4 and later stages, unc-17 fosmid expression (otIs576) becomes apparent in both soma and axon (top panels). The expression of the pan-neuronal marker rab-3 (otIs355) is also first observed in late L4 (bottom panels). (C) Hermaphrodite-specific VC neurons turn on unc-17 and cho-1 only in late L4 (note that cho-1 is NOT in VC4/5); this is later than the onset of the same genes in VA and VB neurons (VA, VB and VC neurons are labeled with the HOX gene lin-39). Transgenes: wgIs18 = lin-39 fosmid reporter; otIs544 = cho-1 fosmid reporter. (D) Sexually dimorphic neurotransmitter identity of a sex-shared neuron class. The AIM neuron expresses cho-1 (and unc-17; not shown) in adult males, but expresses eat-4/VGLUT instead in hermaphrodites Transgenes: otIs354 = cho-1 fosmid reporter; otIs518 = eat-4 fosmid reporter. (E) Sexually dimorphic neurotransmitter switch. Until the L3 stage, both male and hermaphrodite AIM neurons are glutamatergic (express eat-4/VGLUT). While hermaphrodites continue to express eat-4, males downregulate eat-4 and turn on cho-1 (and unc-17; not shown). (F) The neurotransmitter switch is cell-autonomously controlled by the sex-determination pathway. In the upper panels, the masculinizing fem-3 gene is force-expressed in the AIM neurons (with the eat-4prom11 driver) in otherwise hermaphroditic animals; in the lower panels, the masculinizing tra-2 intracellular domain ('tra-2ic') is expressed in AIM neurons of the male. Quantification is provided on the right.
Figure 6 with 1 supplement
Regulatory factors affecting cholinergic identity.
We examined 20 animals for each genotype and for every mutant strain the described phenotype was observed in >80% of animals. (A) The LIM homeobox transcription factor lim-4 is required for unc-17 fosmid reporter expression (left panel) and cho-1 fosmid reporter expression (right panel) in AWB and SMB neurons . AWB neurons were visualized by DiI staining in the unc-17 fosmid reporter expressing strain. AWB and SMB show no fosmid reporter expression in the lim-4 mutant. (B) The Otx-type homeobox transcription factor ceh-14 is required for unc-17 and cho-1 fosmid reporter expression in PVC and unc-17 fosmid reporter expression in PVN. PVC neurons show a decrease in unc-17 and cho-1 fosmid reporter expression in the ceh-14 mutant compared to wild type. PVN neurons show no unc-17 fosmid reporter expression in the ceh-14 mutant. Note that PVN does not express cho-1 fosmid reporter in wild type animals. (C) The homeobox transcription factors unc-30 and lin-11 are required for normal expression of the unc-17 and cho-1 fosmid reporters. Cholinergic identity genes are downregulated in PVP neurons starting at L1 (top panels) and continuing until the L4/adult stage (bottom panels) in unc-30 and lin-11 mutant strains compared to wild type. (D) The homeobox transcription factor unc-42 is required for unc-17 and cho-1 fosmid reporter expression in RIV, SMD, RMD and SIB. (E) The POU homeobox transcription factor unc-86 is required for unc-17 and cho-1 fosmid reporter expression in RIH. (F) A wild type male is shown in the top panel for reference. unc-86 (middle panel) is also required for unc-17 and cho-1 fosmid reporter expression in URX and in the CEM male-specific neurons. In the absence of unc-86 the AIM neurons did not show expression of unc-17 and cho-1 fosmid reporters in the L4/adult male. The LIM homeobox transcription factor ceh-14 is required for the AIM neurons to express unc-17 and cho-1 fosmid reporters in the L4/adult male (bottom panel). Transgenes: otIs576 = unc-17 fosmid reporter; otIs544 = cho-1 fosmid reporter.
Figure 6—figure supplement 1
Continuous expression of transcription factors fosmid reporters in cholinergic neurons.
AIM and PVC were labeled by cho-1 and ceh-14 fosmid reporters. PVN was labeled by ceh-14 fosmid reporter but it did not express cho-1 (see Table 1). ADF and PVP were labeled by cho-1 and lin-11 fosmid reporters. URX, RIR and RIH were labeled by by cho-1 and unc-86 fosmid reporters. PVP was labeled by cho-1 and unc-30 fosmid reporters. AVA, AVE, AVEs, RIV, RMD, SAA, SIB and SMD were labeled by cho-1 and unc-42 fosmid reporters.
Figure 7 with 1 supplement
unc-3 is a circuit-associated transcription factor.
(A) Expression pattern of an unc-3 fosmid-based reporter (otIs591). Overlap with a cho-1 fosmid-based reporter (otIs544) is shown in all panels. The upper panels are the same as the lower, but a Nomarski image has been added for orientation purposes. unc-3 expression was also detected in PDA, PDB and PVP in the pre-anal ganglion (data not shown). (B) The expression of the unc-17 and cho-1 fosmid reporters is downregulated in command interneurons (AVA, AVB, AVD, AVE, PVC) and the tail neuron DVA in unc-3 mutant animals (identical results were obtained using two unc-3 alleles, e151 generates a premature STOP and n3435 is a deletion allele). Quantification is shown on the right. Twenty animals were analyzed at the fourth larval stage (L4) per genotype. Note that the effect of unc-3 on unc-17 expression in the command interneurons (this figure) is not as fully penetrant as it is in VNC motor neurons (Kratsios et al., 2011). (C) Gap junctions that command interneurons make are visualized with gfp tagging the innexin protein UNC-7, as previously described (Starich et al., 2009) (transgene: iwIs47). Dotted white lines delineate the location of the VNC. A significant decrease in the number of the UNC-7::GFP puncta was observed in the VNC of unc-3(n3435) mutant animals (quantification shown on the right with average values and standard deviation). A student’s t test was performed. ***p value <0.0001. (D) Reconstruction of the chemical synapse connectivity of the AVA command interneurons in a wild type and an unc-3(e151/MnH205) mutant animal. Less synaptic input onto AVA neurons and output from the AVA neurons was observed in the unc-3 mutant animal. This is not merely an effect of axonal process misplacement since in unc-3 mutants, AVA processes still run adjacent to the processes of the neurons it normally makes synaptic contacts to. More than 600 electron micrographs were reconstructed per genotype. In square brackets, the location (number of electron micrograph) for each chemical synapse is shown, and the number of consecutive micrographs in which a synapse was detected is also shown in parenthesis.
Figure 7—figure supplement 1
UNC-3 has no effect on glutamate receptor expression in command interneurons.
(A) The expression of PDA identity markers exp-1, ace-3/4, and cog-1 is affected in unc-3 mutant animals. Quantification is provided on the right. For cog-1prom::gfp, n = 25 for wild type and unc-3(e151). For ace-3/-4prom::gfp and exp-1prom::gfp, n = 20 for wild type and unc-3(e151). Fisher’s exact test was performed. **p value <0.01; ***p value < 0.0001. (B) The expression of multiple glutamate receptor genes (nmr-1, nmr-2, glr-1, glr-2, glr-4, glr-5) is unaffected in command interneurons (AVA, AVB, AVD, AVE, PVC) of unc-3 null animals. Similarly, the expression of the ACh receptor subunit encoding gene acr-15 is not affected in the AVA and AVB neurons of unc-3 mutants. Quantification is provided on the right. Number of animals examined = 20 animals per reporter gene per genotype. Moreover, the expression of flp-18 and rig-3 (AVA markers), as well as opt-3 (AVE marker) is not affected in unc-3 mutants (N = 20, data not shown). In addition, the expression of several identity genes (glr-5, glutamate receptor; twk-16, potassium channel; nlp-12, neuropeptide; zig-5, immunoglobulin superfamily gene; ser-2, serotonin receptor) for the DVA interneuron is unaffected in unc-3 mutants (data not shown).
Figure 8
Coupling of cholinergic identity with other identity features.
(A) An mgl-1 reporter transgene does not show expression in RMD neurons in the absence of unc-42. (B) In the absence of ceh-14 the AIM neurons do not show eat-4 fosmid reporter and flp-10 reporter expression. 5-HT staining is not detectable in AIM neurons in the ceh-14 mutant. In the absence of ceh-14 the PVC neurons do not show nmr-1 or glr-1 reporter expression. Number of animals examined = 20 animals per reporter gene per genotype. (C) The expression of PDA identity markers exp-1, ace-3/4, and cog-1 is lost in unc-3 mutant animals. For cog-1prom::gfp, 25 of 25 wild-type and 1 of 25 unc-3(e151) animals showed cog-1prom::gfp expression in PDA. For ace-3/-4prom::gfp, 20 of 20 wild-type and 0 of 20 unc-3(e151) animals showed ace-3/-4prom::gfp expression in PDA. For exp-1prom::gfp, 20 of 20 wild-type and 11 of 20 unc-3(e151) animals showed exp-1prom::gfp expression in PDA.
Figure 9
Circuit-associated transcription factors.
(A) Ventral cord motor circuit as shown in White et al. (1986), but now superimposed with neurotransmitter identity and expression pattern of the unc-3 transcription factor. unc-3 controls the cholinergic identity of every single neuron in this circuit. Next to the circuit diagram, a number of different regulated mutual 3-neuron networks motifs are shown. These motifs are either embedded in the circuit and provide a connection to neurons located outside the circuit (e.g. glutamatergic sensory neurons). In all cases unc-3 controls cholinergic identity of the mutually connected command interneurons ('CI') and in those cases where the mutually connected neurons receive cholinergic interneuron input, unc-3 controls the identity of the entire microcircuit. (B) unc-42 controls the cholinergic identity of interconnected head motor neurons, and glutamatergic signaling between ASH sensory neurons (whose glutamatergic identity is controlled by unc-42 (Serrano-Saiz et al., 2013) and cross-connected command interneurons in which unc-42 controls glutamate receptor expression (Brockie et al., 2001) (shown in Table 4). Red boxes indicate the neurons affected by the indicated transcription factor.
Figure 10
A single UNC-3 binding site is required for cho-1 expression in all distinct unc-3-dependent cholinergic neuron types.
(A) Schematic showing of the cho-1 locus and the location of the UNC-3 binding site (COE motif) relative to ATG for the fosmid reporters and 280bp promoter fusion. (B, C) A cho-1 fosmid reporter (~28 kb) that contains an intact COE motif shows expression in all cholinergic neurons including the ventral nerve cord (VNC) motor neurons (MNs), the command interneurons (AVA, AVB, AVD, AVE, PVC), and the interneuron DVA. Mutation of the COE motif in the context of this cho-1 fosmid-based reporter results in selective loss of reporter gene expression in VNC MNs residing at the retrovesicular ganglion and all command interneurons (only AVA and AVE head interneurons are shown). (B) cho-1 fosmid reporter versus cho-1_COEmut fosmid reporter in an adult head. (C) cho-1 fosmid reporter versus cho-1_COEmut fosmid reporter in an adult tail. Reporter gene expression is also lost in tail neurons DVA and PVC. The transgenic line rab-3prom::rfp drives reporter gene expression in the entire nervous system and was used in the background to facilitate neuronal identification. (D) A short fragment (280 bp) of the cho-1 cis-regulatory region containing the COE motif is sufficient to drive reporter gene expression only in VNC MNs. This fragment does not show expression in command interneurons located at the head and tail of the animal. (E) A short fragment (250 bp) of the unc-17 cis-regulatory region containing the COE motif is sufficient to drive reporter gene expression only in VNC MNs.
Videos
Video 1
Cholinergic and glutamatergic head neurons.
Confocal image stack of a transgenic worm expressing cho-1::mChopti (otIs544) and eat-4::yfp (otIs388) fosmid reporter gene constructs in the head.
Tables
Table 1
Cholinergic neurons in the hermaphrodite.
| Neuron type | Neuron class | VAChT/ChAT1 | ChT 2 | AChE 3 | Co-transmitter | Previous ID 4 |
|---|---|---|---|---|---|---|
| Sensory neuron (9 classes)
| ADF L/R | ++ | ++ |
| Serotonin | no |
| ALN L/R | ++ | ++ |
|
| yes 5 | |
| ASJ L/R | + | ++ |
|
| no | |
| AWB L/R | ++ | ++ |
| no | ||
| IL2 D/V L/R | +++ | +++ | ace-3/4 |
| yes 6 | |
| PLN L/R | ++ | ++ |
| yes 5 | ||
| URA D/V L/R | +++ | ++ | ace-3/4 |
| yes 6 | |
| URB L/R | ++ | + | ace-3/4 |
| yes 6 | |
| URX L/R | ++ | ++ | ace-3/4 |
| no | |
| Interneuron (19 classes) | AIA L/R | +++ | ++ | ace-3/4 |
| yes 7 |
| AIN L/R | ++ | +++ |
| yes 6 | ||
| AIY L/R | +++ | +++ |
|
| yes 7 | |
| AVA L/R | +++ | ++ | ace-2 |
| no | |
| AVB L/R | +++ | ++ | ace-2 |
| no | |
| AVD L/R | +++ | ++ | ace-2 |
| no | |
| AVE L/R | +++ | ++ | ace-2 |
| no | |
| AVG | + |
| no | |||
| DVA | +++ | +++ | ace-2; ace-3/4 |
| no 8 | |
| PVC L/R | +++ | + |
| yes 5 | ||
| PVN L/R | ++ |
| no | |||
| PVP L/R | +++ | ++ |
| yes 5 | ||
| RIB L/R | (+)* | +++ |
| no | ||
| RIF L/R | ++ | ++ |
|
| no | |
| RIH | +++ | ++ | ace-2; ace-3/4 | Serotonin | no | |
| RIR | ++ | ++ | no | |||
| RIV L/R | ++ | ++ | ace-3/4 | no | ||
| SAA D/V L/R | ++ |
|
| no | ||
| SDQ L/R | ++ | ++ |
|
| yes 5 | |
| Motor neuron (17 classes) | AS1-11 | ++ | ++ | ace-2 |
| yes 5 |
| DA1-9 | ++ | ++ | ace-2 |
| yes 5 | |
| DB1-7 | ++ | ++ | ace-2 |
| yes 5 | |
| HSN L/R | ++ |
| Serotonin | yes 5 | ||
| PDA | ++ | ++ | ace-2; ace-3/4 |
| no | |
| PDB | ++ | ++ |
| no | ||
| RMD D/V L/R | +++ | +++ | ace-3/4 |
| yes 5 | |
| RMF L/R | ++ | ++ |
| no | ||
| RMH L/R | ++ | ++ |
| no | ||
| SAB D V L/R | ++ | ++ |
| yes 9 | ||
| SIA D/V L/R | +++ | ++ | ace-3/4 |
| no | |
| SIB D/V L/R | +++ | ++ |
|
| no | |
| SMB D/V L/R | +++ | ++ |
|
| no | |
| SMD D/V L/R | +++ | +++ | ace-3/4 |
| no 10 | |
| Motor neuron (17 classes) | VA1-12 | ++ | ++ | ace-2 |
| yes 5 |
| VB1-11 | ++ | ++ | ace-2 |
| yes 5 | |
| VC1-3 VC6 | ++ | ++ |
|
| yes 5 | |
| VC4-5 | ++ |
|
| Serotonin | yes 5 | |
| Pharyngeal |
|
|
|
|
|
| Polymodal (7 classes) | I1 L/R | ++ |
|
|
| no |
| I3 | ++ |
|
|
| no | |
| MC L/R | ++ |
|
|
| yes 11 | |
| M1 | ++ |
|
|
| no | |
| M2 L/R | ++ |
|
|
| no | |
| M4 | +++ | +++ | ace-2 |
| no | |
| M5 | +++ | +++ |
|
| no | |
| unc-17(+): 52 classes, 159 neurons |
|
|
|
|
|
-
*Expression of cho-1 in the RIB neurons is strong but unc-17 expression is, at best, very dim.
-
1Gray shading indicates unc-17/cha-1 (VAChT/ChAT) expression as assessed by fosmid reporter and antibody staining.
-
2Gray shading indicates cho-1 (ChT) expression as assessed by fosmid reporters.
-
3Gray shading indicates reporters expression of one of the C. elegans ace (AChE) genes.
-
4Previously identified as a cholinergic neuron: 'yes' – see indicated references. 'no' - newly identified in this study. Only published data is considered, personal communications in Rand and Nonet (1997) were not taken into consideration.
-
8Previously proposed to be DVC (Duerr et al., 2008) but based on position and markers reassigned to DVA.
-
10Based on our identification as SMB as cholinergic, Kim et al. (2015) demonstrated that lim-4 controls SMB cholinergic identity (see also Table 6).
Table 2
Neurotransmitter map of the hermaphrodite nervous system.
| Neuron class | Neuron | Neurotransmitter | Notes |
|---|---|---|---|
| ADA | ADAL | Glu | |
| ADAR | Glu | ||
| ADE | ADEL | DA | |
| ADER | DA | ||
| ADF | ADFL | ACh & 5HT | |
| ADFR | ACh & 5HT | ||
| ADL | ADLL | Glu | |
| ADLR | Glu | ||
| AFD | AFDL | Glu | |
| AFDR | Glu | ||
| AIA | AIAL | ACh | |
| AIAR | ACh | ||
| AIB | AIBL | Glu | |
| AIBR | Glu | ||
| AIM | AIML | Glu & 5HT | |
| AIMR | Glu & 5HT | ||
| AIN | AINL | ACh | |
| AINR | ACh | ||
| AIY | AIYL | ACh | |
| AIYR | ACh | ||
| AIZ | AIZL | Glu | |
| AIZR | Glu | ||
| ALA | ALA | Unknown (orphan) | Newly assigned as mechanosensory (based on Sanders et al., 2013) |
| ALM | ALML | Glu | |
| ALMR | Glu | ||
| ALN | ALNL | ACh | Classified as sensory because of expression of oxygen sensors |
| ALNR | ACh | ||
| AQR | AQR | Glu | |
| AS | AS1 | ACh | |
| AS2 | ACh | ||
| AS3 | ACh | ||
| AS4 | ACh | ||
| AS5 | ACh | ||
| AS6 | ACh | ||
| AS7 | ACh | ||
| AS8 | ACh | ||
| AS9 | ACh | ||
| AS10 | ACh | ||
| AS11 | ACh | ||
| ASE | ASEL | Glu | |
| ASER | Glu | ||
| ASG | ASGL | Glu | |
| ASGR | Glu | ||
| ASH | ASHL | Glu | |
| ASHR | Glu | ||
| ASI | ASIL | Unknown (orphan) | |
| ASIR | Unknown (orphan) | ||
| ASJ | ASJL | ACh | |
| ASJR | ACh | ||
| ASK | ASKL | Glu | |
| ASKR | Glu | ||
| AUA | AUAL | Glu | |
| AUAR | Glu | ||
| AVA | AVAL | ACh | |
| AVAR | ACh | ||
| AVB | AVBL | ACh | |
| AVBR | ACh | ||
| AVD | AVDL | ACh | |
| AVDR | ACh | ||
| AVE | AVEL | ACh | |
| AVER | ACh | ||
| AVF | AVFL | Unknown (orphan) | |
| AVFR | Unknown (orphan) | ||
| AVG | AVG | ACh | |
| AVH | AVHL | Unknown (orphan) | |
| AVHR | Unknown (orphan) | ||
| AVJ | AVJL | Unknown (orphan) | |
| AVJR | Unknown (orphan) | ||
| AVK | AVKL | Unknown (orphan) | |
| AVKR | Unknown (orphan) | ||
| AVL | AVL | GABA | |
| AVM | AVM | Glu | |
| AWA | AWAL | Unknown (orphan) | |
| AWAR | Unknown (orphan) | ||
| AWB | AWBL | ACh | |
| AWBR | ACh | ||
| AWC | AWCL | Glu | |
| AWCR | Glu | ||
| BAG | BAGL | Glu | |
| BAGR | Glu | ||
| BDU | BDUL | Unknown (orphan) | |
| BDUR | Unknown (orphan) | ||
| CAN | CANL | unknown MA (cat-1) | |
| CANR | unknown MA (cat-1) | ||
| CEP | CEPDL | DA | |
| CEPDR | DA | ||
| CEPVL | DA | ||
| CEPVR | DA | ||
| DA | DA1 | ACh | |
| DA2 | ACh | ||
| DA3 | ACh | ||
| DA4 | ACh | ||
| DA5 | ACh | ||
| DA6 | ACh | ||
| DA7 | ACh | ||
| DA8 | ACh | ||
| DA9 | ACh | ||
| DB | DB1/3 | ACh | |
| DB2 | ACh | ||
| DB3/1 | ACh | ||
| DB4 | ACh | ||
| DB5 | ACh | ||
| DB6 | ACh | ||
| DB7 | ACh | ||
| DD | DD1 | GABA | |
| DD2 | GABA | ||
| DD3 | GABA | ||
| DD4 | GABA | ||
| DD5 | GABA | ||
| DD6 | GABA | ||
| DVA | DVA | ACh | |
| DVB | DVB | GABA | |
| DVC | DVC | Glu | |
| FLP | FLPL | Glu | |
| FLPR | Glu | ||
| HSN | HSNL | ACh & 5HT | |
| HSNR | ACh & 5HT | ||
| IL1 | IL1DL | Glu | Also a clear motor neuron |
| IL1DR | Glu | ||
| IL1L | Glu | ||
| IL1R | Glu | ||
| IL1VL | Glu | ||
| IL1VR | Glu | ||
| IL2 | IL2DL | ACh | Also a clear motor neuron |
| IL2DR | ACh | ||
| IL2L | ACh | ||
| IL2R | ACh | ||
| IL2VL | ACh | ||
| IL2VR | ACh | ||
| LUA | LUAL | Glu | |
| LUAR | Glu | ||
| OLL | OLLL | Glu | |
| OLLR | Glu | ||
| OLQ | OLQDL | Glu | |
| OLQDR | Glu | ||
| OLQVL | Glu | ||
| OLQVR | Glu | ||
| PDA | PDA | ACh | |
| PDB | PDB | ACh | |
| PDE | PDEL | DA | |
| PDER | DA | ||
| PHA | PHAL | Glu | |
| PHAR | Glu | ||
| PHB | PHBL | Glu | |
| PHBR | Glu | ||
| PHC | PHCL | Glu | |
| PHCR | Glu | ||
| PLM | PLML | Glu | |
| PLMR | Glu | ||
| PLN | PLNL | ACh | |
| PLNR | ACh | ||
| PQR | PQR | Glu | |
| PVC | PVCL | ACh | |
| PVCR | ACh | ||
| PVD | PVDL | Glu | |
| PVDR | Glu | ||
| PVM | PVM | Unknown (orphan) | |
| PVN | PVNL | ACh | Only very few minor NMJs, more prominent neuron-neuron synapses |
| PVNR | ACh | ||
| PVP | PVPL | ACh | |
| PVPR | ACh | ||
| PVQ | PVQL | Glu | |
| PVQR | Glu | ||
| PVR | PVR | Glu | |
| PVT | PVT | Unknown (orphan) | |
| PVW | PVWL | Unknown (orphan) | |
| PVWR | Unknown (orphan) | ||
| RIA | RIAL | Glu | |
| RIAR | Glu | ||
| RIB | RIBL | ACh | |
| RIBR | ACh | ||
| RIC | RICL | Octopamine | |
| RICR | Octopamine | ||
| RID | RID | Unknown (orphan) | |
| RIF | RIFL | ACh | |
| RIFR | ACh | ||
| RIG | RIGL | Glu | |
| RIGR | Glu | ||
| RIH | RIH | ACh & 5HT | |
| RIM | RIML | Glu & Tyramine | |
| RIMR | Glu & Tyramine | ||
| RIP | RIPL | Unknown (orphan) | |
| RIPR | Unknown (orphan) | ||
| RIR | RIR | ACh | |
| RIS | RIS | GABA | |
| RIV | RIVL | ACh | Only very few minor NMJs, more prominent neuron-neuron synapses |
| RIVR | ACh | ||
| RMD | RMDDL | ACh | |
| RMDDR | ACh | ||
| RMDL | ACh | ||
| RMDR | ACh | ||
| RMDVL | ACh | ||
| RMDVR | ACh | ||
| RME | RMED | GABA | |
| RMEL | GABA | ||
| RMER | GABA | ||
| RMEV | GABA | ||
| RMF | RMFL | ACh | |
| RMFR | ACh | ||
| RMG | RMGL | Unknown (orphan) | |
| RMGR | Unknown (orphan) | ||
| RMH | RMHL | ACh | |
| RMHR | ACh | ||
| SAA | SAADL | ACh | |
| SAADR | ACh | ||
| SAAVL | ACh | ||
| SAAVR | ACh | ||
| SAB | SABD | ACh | Makes clear neuromuscular junctions |
| SABVL | ACh | ||
| SABVR | ACh | ||
| SDQ | SDQL | ACh | |
| SDQR | ACh | ||
| SIA | SIADL | ACh | Makes clear neuromuscular junctions |
| SIADR | ACh | ||
| SIAVL | ACh | ||
| SIAVR | ACh | ||
| SIB | SIBDL | ACh | Makes clear neuromuscular junctions |
| SIBDR | ACh | ||
| SIBVL | ACh | ||
| SIBVR | ACh | ||
| SMB | SMBDL | ACh | |
| SMBDR | ACh | ||
| SMBVL | ACh | ||
| SMBVR | ACh | ||
| SMD | SMDDL | ACh | |
| SMDDR | ACh | ||
| SMDVL | ACh | ||
| SMDVR | ACh | ||
| URA | URADL | ACh | Also a clear motor neuron |
| URADR | ACh | ||
| URAVL | ACh | ||
| URAVR | ACh | ||
| URB | URBL | ACh | |
| URBR | ACh | ||
| URX | URXL | ACh | |
| URXR | ACh | ||
| URY | URYDL | Glu | |
| URYDR | Glu | ||
| URYVL | Glu | ||
| URYVR | Glu | ||
| VA | VA1 | ACh | |
| VA2 | ACh | ||
| VA3 | ACh | ||
| VA4 | ACh | ||
| VA5 | ACh | ||
| VA6 | ACh | ||
| VA7 | ACh | ||
| VA8 | ACh | ||
| VA9 | ACh | ||
| VA10 | ACh | ||
| VA11 | ACh | ||
| VA12 | ACh | ||
| VB | VB1 | ACh | |
| VB2 | ACh | ||
| VB3 | ACh | ||
| VB4 | ACh | ||
| VB5 | ACh | ||
| VB6 | ACh | ||
| VB7 | ACh | ||
| VB8 | ACh | ||
| VB9 | ACh | ||
| VB10 | ACh | ||
| VB11 | ACh | ||
| VC | VC1 | ACh | |
| VC2 | ACh | ||
| VC3 | ACh | ||
| VC4 | ACh & 5HT | ||
| VC5 | ACh & 5HT | ||
| VC6 | ACh | ||
| VD | VD1 | GABA | |
| VD2 | GABA | ||
| VD3 | GABA | ||
| VD4 | GABA | ||
| VD5 | GABA | ||
| VD6 | GABA | ||
| VD7 | GABA | ||
| VD8 | GABA | ||
| VD9 | GABA | ||
| VD10 | GABA | ||
| VD11 | GABA | ||
| VD12 | GABA | ||
| VD13 | GABA | ||
| Summary for extrapharyngeal neurons | |||
| Sensory neuron: | Sensory neuron: | ||
| 38/104 classes | ACh: 9 classes | ||
| 87/282 total neurons | Glu: 22 | ||
| Motor neuron: | GABA: 0 | ||
| 24/104 | Aminergic: 3 (all Dopa) | ||
| 118/282 | Unknown: 4 (ASI, AWA, PVM, ALA) | ||
| Interneuron | Motor neuron: | ||
| 42/104 | ACh: 17 classes | ||
| 77/282 | Glu: 1 (RIM) | ||
| GABA: 5 | |||
| Aminergic: 0 | |||
| Unknown: 1 (RMG) | |||
| Interneuron: | |||
| ACh: 19 classes | |||
| Glu: 11 | |||
| GABA: 1 (RIS) | |||
| Aminergic: 2 (CAN, RIC) | |||
| Unknown: 9 | |||
| Pharyngeal neurons | |||
| I1 | I1L | ACh | Due to connectivity and rudimentary sensory endings, all polymodal |
| I1R | ACh | ||
| I2 | I2L | Glu | |
| I2R | Glu | ||
| I3 | I3 | ACh | |
| I4 | I4 | Unknown (orphan) | |
| I5 | I5 | Glu & 5HT | |
| I6 | I6 | Unknown (orphan) | |
| M1 | M1 | ACh | |
| M2 | M2L | ACh | |
| M2R | ACh | ||
| M3 | M3L | Glu | |
| M3R | Glu | ||
| M4 | M4 | ACh | |
| M5 | M5 | ACh | |
| MC | MCL | ACh | |
| MCR | ACh | ||
| MI | MI | Glu | |
| NSM | NSML | 5HT | |
| NSMR | 5HT | ||
Table 3
Neurons receiving cholinergic inputs. Includes pharyngeal neurons. Data from www.wormwiring.org.
| Connectivity * | Neuron class | # | |
|---|---|---|---|
| Receiving ACh input | Cholinergic neurons | ADF, AIA, AIN, AIY, ALN, AS, ASJ, AVA, AVB, AVD, AVE, AWB, DA, DB, DVA, I3, IL2, M2, M4, PLN, PVC, PVN, PVP, RIB, RIF, RIH, RIR, RIV, RMD, RMF, RMH, SAA, SAB, SDQ, SIA, SIB, SMB, SMD, URA, URB, URX, VA, VB, VC | 44 |
| Non-cholinergic neurons | ADA, ADE, ADL, AFD, AIB, AIM, AIZ, ALA, ALM, AQR, ASE, ASG, ASH, ASI, ASK, AUA, AVF, AVH, AVJ, AVK, AVL, ASA, AWC, BAG, BDU, CEP, DD, DVC, I2, I4, I5, IL1, LUA, M1, M3, MC, NSM, OLL, OLQ, PQR, PVQ, PVR, PVT, PVW, RIA, RIC, RID, RIG, RIM, RIP, RIR, RIS, RME, RMG, URY, VD | 56 | |
| Receiving no ACh input | Cholinergic neuron | AVG, HSN, I1, M5, PDA, PDB | 6 |
| Innervate cholinergic neuron | AVM, DVB, FLP, I6, MI, PDE, PHA, PHB, PHC, PLM, PVD, PVM | 12 | |
| Neither of the above | CAN | 1 | |
Table 4
Occurences of the 'Regulated Mutual' network motif.
| Type 1: Sensory>command interneurons | Type 2: Interneurons>command interneurons | Type 3: Sensory neurons>head motor neurons | Type 4: Interneurons>head motor neurons | Type 5: Egg laying circuit | Miscellaneous | |||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| SN | CI | CI | opp. | IN | CI | CI | opp. | SN | hMN | hMN | IN | hMN | hMN | |||||||
| ADEL | AVAL | AVAR | ADAL | AVAL | AVAR | CEPVL | RMDDL | RMDVR | RIAL | RMDDL | RMDVR | AIML | AVFL | AVFR | PHBL | VA12 | AVAL | |||
| ADER | AVAL | AVAR | ADAL | AVAR | AVBL | yes | IL1DL | RMDDR | RMDVL | RIAL | RMDDR | RMDVL | AIMR | RIFR | HSNR | PHCL | VA12 | AVAL | ||
| ADER | AVAR | AVDR | ADAL | AVAR | AVBR | yes | IL1DR | RMDDL | RMDVR | RIAR | RMDDL | RMDVR | AIMR | AVFL | AVFR | VA12 | PVCL | PVCR | ||
| ADER | AVAR | AVER | ADAR | AVAR | AVBL | yes | IL1L | RMDDL | RMDVR | RIAR | RMDDR | RMDVL | AIMR | AVFL | HSNR | AVEL | DA01 | AS01 | ||
| ADLL | AVAL | AVAR | ADAR | AVAR | AVBR | yes | IL1L | RMDL | RMDR | RICR | SMDDL | SMDVR | AIMR | AVFR | HSNL | AVER | DA01 | AS01 | ||
| ADLL | AVAL | AVDL | ADAR | AVAR | AVDR | IL1R | RMDDR | RMDVL | RICR | SMDDR | SMDVL | HSNL | AVFL | HSNR | AVHL | ADFR | AWBR | |||
| ADLL | AVAR | AVBL | yes | ALA | AVAR | AVER | IL1R | RMDL | RMDR | RIML | SMDDR | SMDVL | AWAR | ADFR | AWBR | |||||
| ADLL | AVAR | AVDR | AUAR | AVAR | AVER | IL1VL | RMDDL | RMDVR | RIMR | RMDL | RMDR | CEPVR | IL2VR | URAVR | ||||||
| ADLR | AVAR | AVBL | AVBR | AVAL | AVDL | IL1VR | RMDDR | RMDVL | RIMR | SMDDL | SMDVR | |||||||||
| ADLR | AVAR | AVBR | AVDL | AVAR | AVDR | IL2L | RMDL | RMDR | RIS | RMDL | RMDR | |||||||||
| ADLR | AVAR | AVDR | AVEL | AVAL | AVAR | OLLL | SMDDL | SMDVR | RIVR | SMDDL | SMDVR | |||||||||
| ADLR | AVAR | PVCL | yes | AVER | AVAL | AVDL | OLLR | SMDDL | SMDVR | RMGR | RMDL | RMDR | ||||||||
| ALML | PVCL | PVCR | yes | AVER | AVDL | AVEL | URYDL | RMDDR | RMDVL | |||||||||||
| AQR | AVAL | AVAR | AVG | AVAL | AVAR | URYDR | RMDDL | RMDVR | unc-42 | unc-42 | ||||||||||
| AQR | AVAL | AVDL | AVG | AVAR | AVBL | yes | URYDR | SMDDL | SMDVR | |||||||||||
| AQR | AVAL | PVCR | yes | AVG | AVAR | AVBR | yes | URYVL | RMDDL | RMDVR | ||||||||||
| AQR | AVAR | AVBL | yes | AVG | AVAR | AVDR | URYVR | RMDDR | RMDVL | |||||||||||
| AQR | AVAR | AVBR | yes | AVJR | AVAR | AVBL | yes | |||||||||||||
| AQR | AVAR | PVCR | yes | AVJR | AVAR | AVDR | unc-42 | unc-42 | ||||||||||||
| ASHL | AVAL | AVDL | AVJR | AVAR | AVER | |||||||||||||||
| ASHR | AVAR | AVBR | yes | AVJR | AVAR | PVCL | yes | |||||||||||||
| ASHR | AVAR | AVER | AVJR | AVAR | PVCR | yes | ||||||||||||||
| ASHR | AVAR | PVCL | AVJR | PVCL | PVCR | yes | ||||||||||||||
| AVM | PVCL | PVCR | BDUR | AVAL | PVCL | yes | ||||||||||||||
| BAGL | AVAR | AVER | DVA | AVAL | PVCL | yes | ||||||||||||||
| FLPL | AVAL | AVAR | DVC | AVAL | AVAR | |||||||||||||||
| FLPL | AVAL | AVDL | LUAR | AVAL | AVDL | |||||||||||||||
| FLPL | AVAL | PVCR | yes | LUAR | AVAL | PVCR | yes | |||||||||||||
| FLPL | AVAR | AVBL | yes | PVCR | AVDL | AVEL | ||||||||||||||
| FLPL | AVAR | AVBR | yes | PVNL | AVAL | AVDL | ||||||||||||||
| FLPL | AVAR | AVDR | PVNL | AVAL | PVCL | yes | ||||||||||||||
| FLPL | AVAR | PVCR | yes | PVPL | AVAL | AVAR | ||||||||||||||
| FLPR | AVAL | AVAR | PVPL | AVAL | PVCL | yes | ||||||||||||||
| FLPR | AVAL | AVDL | PVPL | AVAL | PVCR | yes | ||||||||||||||
| FLPR | AVAR | AVBL | yes | PVPL | AVAR | AVBL | yes | |||||||||||||
| FLPR | AVAR | AVBR | yes | PVPL | AVAR | AVBR | yes | |||||||||||||
| FLPR | AVAR | AVDR | PVPL | AVAR | AVDR | |||||||||||||||
| FLPR | AVAR | AVER | PVPL | AVAR | PVCL | yes | ||||||||||||||
| FLPR | AVDL | AVEL | PVPL | AVAR | PVCR | yes | ||||||||||||||
| PHBL | AVAL | AVAR | PVPL | PVCL | PVCR | |||||||||||||||
| PHBL | AVAL | AVDL | PVPR | AVAR | AVBR | yes | ||||||||||||||
| PHBL | AVAL | PVCL | yes | PVPR | AVAR | PVCL | yes | |||||||||||||
| PHBL | AVAR | PVCL | yes | PVPR | AVAR | PVCR | yes | |||||||||||||
| PHBR | AVAL | AVAR | PVPR | PVCL | PVCR | |||||||||||||||
| PHBR | AVAL | AVDL | RIBR | AVAR | AVER | |||||||||||||||
| PHBR | AVAL | PVCL | yes | RICL | AVAL | AVAR | ||||||||||||||
| PHBR | AVAL | PVCR | yes | RICR | AVAL | AVAR | ||||||||||||||
| PHBR | AVAR | AVDR | SDQL | AVAL | AVAR | |||||||||||||||
| PHBR | AVAR | PVCL | yes | SDQL | AVAL | AVDL | ||||||||||||||
| PHBR | AVAR | PVCR | yes | |||||||||||||||||
| PHBR | PVCL | PVCR | unc-3 | unc-3 | ||||||||||||||||
| PHCL | AVAL | PVCL | yes | |||||||||||||||||
| PQR | AVAL | AVAR | ||||||||||||||||||
| PQR | AVAL | AVDL | ||||||||||||||||||
| unc-3 | unc-3 | |||||||||||||||||||
-
Yellow: Glu, red: ACh, green: Aminergic, blue: GABA.
-
opp.: command interneurons control opposite drives (forward/reverse).
-
SN: sensory neuron, IN: interneuron, CI: command interneuron, hMN: head motor neuron. Black bar: Transcription factor controlling cholinergic identity. Note that most interconnected neurons are controlled by the same transcription factor.
Table 5
Male-specific cholinergic neurons.
| Neuron type | Neuron class | unc-17 fosmid expression | cho-1 fosmid expression | Co-transmitter | Previous ID |
|---|---|---|---|---|---|
| Sensory neuron (7 classes) | CEM D/V L/R | ++ | ++ | no | |
| R1A, R2A, R3A, R4A, R6A | ++ | ++ | yes 1 | ||
| SPC L/R | ++ | ++ | yes 2 | ||
| SPV L/R | ++ | ++ | yes 3 | ||
| HOB | +++ | ++ | no | ||
| PCB L/R | ++ | ++ | yes 2 | ||
| PCC L/R | ++ | ++ | no | ||
| Interneuron (6 classes) | DVE | ++ |
| no | |
| DVF | ++ | no | |||
| PDC | ++ | ++ | PDC or PGA are also serotonergic 4 | no | |
| PGA | ++ |
| PDC or PGA are also serotonergic 4 | no | |
| PVY | +++ | ++ | yes3 | ||
| PVX | +++ | ++ | yes3 | ||
| Motor neuron (3 classes) | PVZ | +++ | ++ | no | |
| PVV | +++ | ++ | Glutamatergic6 | no | |
| CA1-9* | ++ | ++ | no7 |
-
*CA7-9 do not express cho-1 and have lower levels of unc-17 than CA1-6.
-
6Our unpublished data.
-
7Rand and Nonet cite unpublished observations of cholinergic identity of four CA neurons (Rand and Nonet, 1997). We observe expression of unc-17 in all nine CA neurons (albeit lower in CA7-9).
Table 6
Newly identified transcriptional regulators of cholinergic identity.
| Gene* | DNA binding domain | Neuron class | Effect on identity features | Other neurotransmitter identities affected (neuron class) | ||
Cholinergic identity** | Other identity features** | |||||
unc-17 cha-1 | cho-1 | |||||
| unc-3 (EBF) | Zn finger | PDA | yes | yes | yes | |
| PDB | yes | yes | n.d. | |||
| DVA | yes | yes | no | |||
| PVC | yes | yes | no | |||
| AVA | yes | yes | no | |||
| AVB | yes | yes | no | |||
| AVD | yes | yes | no | |||
| AVE | yes | yes | no | |||
| PVN | yes | n.a. | n.d. | |||
| unc-42 (Prd-type) | Homeodomain | RIV | yes | yes | n.d. | Glu(ASH) 7 |
| RMD | yes | yes | yes | |||
| SMD | yes | yes | n.d. | |||
| SIB | yes | yes | n.d. | |||
| AVA | no | no | yes1 | |||
| AVD | no | no | yes1 | |||
| AVE | no | no | yes1 | |||
| lim-4(Lhx6/8) | Homeodomain | AWB | yes | yes | yes2 | |
| SMB | yes | yes | yes3 | |||
| RIV | no | no | n.d. | |||
| lin-11 (Lhx1) | Homeodomain | ADF | no | no | no | Glu (ASG, ADL) 7 |
| PVP | yes | yes | yes4 | |||
| unc-30 (Pitx) | Homeodomain | PVP | yes | yes | yes4 | GABA (DD, VD) 9 |
| RIH | no | no | n.d. | |||
| unc-86 (Brn3) | Homeodomain | CEM (male) | yes | yes | yes5 | Glu (ALM, PLM, AIM, AIZ, AQR, PQR, PVR) 7 |
| URX | yes | yes | yes6 | |||
| AIM (male) | yes | yes | yes7 | |||
| RIH | yes | yes | yes8 | |||
| ceh-14 (Lhx3/4) | Homeodomain | AIM (male) | yes | yes | yes | Glu (AFD, DVC, PHA, PHB, PHC) 7 |
| PVN | yes | yes | n.d. | |||
| PVC | yes | yes | yes | |||
-
*Vertebrate orthologs in parenthesis. All neuron classes listed express the respective transcription factor tested.
-
**'yes' = expression is downregulated or completely absent; 'no' = no readily observable effect; 'n.d.' = not determined; 'n.a. = not applicable because gene is not expressed in this cell. For primary data see Figure 6, and Figure 8. For data on 'other markers' (≥2 markers tested), see individual footnotes (this data is partly our own data, partly previously reported data). .Previously identified regulators of cholinergic identity are: unc-3 in A-, B-type, AS and SAB motor neurons, unc-86 in IL2, URA, URB, cfi-1 in IL2, URA, ttx-3 in AIY and AIA and ceh-10 in AIY (Altun-Gultekin et al., 2001; Kratsios et al., 2011, 2015; Wenick and Hobert, 2004; Zhang et al., 2014).
Table 7
unc-3 affects the differentiation of a broad set of cholinergic neuron types. nmr and glr genes encode glutamate receptors and expression of neither is affected by unc-3; many of them are instead regulated by either the unc-42, fax-1 or cfi-1, as summarized in Table 8.
| unc-3 (+) neurons | Cholinergic identity in unc-3(-) animals 1 | Other identity features that are NOT affected in unc-3(-) animals 1 | Other identity feature that are affected in unc-3(-) animals | ||
|---|---|---|---|---|---|
| INTER | Command inter- neurons | AVA | unc-17, cho-1 affected | nmr-1, nmr-2, glr-1, glr-2, glr-4, glr-5, acr-15, rig-3, flp-18 | |
| AVB | unc-17, cho-1 affected | acr-15 | |||
| AVD | unc-17, cho-1 affected | nmr-1, nmr-2, glr-1, glr-2, glr-5 | |||
| AVE | unc-17, cho-1 affected | nmr-1, nmr-2, glr-1, glr-2, glr-5, opt-3 | |||
| PVC | unc-17, cho-1 affected | nmr-1, nmr-2, glr-1, glr-2, glr-5 | |||
| Other inter- neurons | DVA | unc-17, cho-1 affected | glr-4, glr-5, twk-16, nlp-12, zig-5, ser-2 | ||
| SAA | unc-17 NOT affected2 | ||||
| PVP | unc-17, cho-1 NOT affected | ||||
MOTOR | Head MNs | SAB | unc-17, cho-1 affected3 | 8/8 markers tested3 | |
| VNC MNs | A,B,AS | unc-17, cho-1 affected3 | 29/34 markers tested3 | ||
| Tail MNs | PDA | unc-17, cho-1 affected | exp-1, ace-3/4, cog-1 1 | ||
| PDB | unc-17, cho-1 affected | ||||
| PVN | unc-17 affected2 |
-
2Cho-1 not expressed in these neurons.
-
3As previously reported by Kratsios et al. (2011), (2015).
Table 8
Transcription factors affecting command interneuron differentiation.
TF | AVA | AVB | AVD | AVE | PVC |
| unc-3 | |||||
| unc-42 | |||||
| fax-1 | |||||
| cfi-1 | |||||
| ceh-14 | |||||
| ACh | |||||
| unc-17 & cho-1 | unc-3 effect | unc-3 effect | unc-3 effect | unc-3 effect | unc-3 effect |
| unc-42 NO effect | unc-42 NO effect | unc-42 NO effect | |||
| ceh-14 effect | |||||
| Other | |||||
| nmr-1 (GluR) | unc-3 NO effect | unc-3 NO effect | unc-3 NO effect | unc-3 NO effect | |
| unc-42 NO effect | unc-42 NO effect | unc-42 NO effect | ceh-14 effect | ||
| fax-1 effect | fax-1 NO effect | fax-1 effect | |||
| cfi-1 effect | cfi-1 effect | ||||
| nmr-2 (GluR) | unc-3 NO effect | unc-3 NO effect | unc-3 NO effect | unc-3 NO effect | |
| unc-42 NO effect | unc-42 NO effect | unc-42 NO effect | | ||
| fax-1 effect | fax-1 NO effect | fax-1 effect | |||
| glr-1 (GluR) | unc-3 NO effect | unc-3 NO effect | unc-3 NO effect | unc-3 NO effect | unc-3 NO effect |
| unc-42 effect | unc-42 NO effect | unc-42 effect | unc-42 effect | ceh-14 effect | |
| fax-1 no effect | fax-1 NO effect | fax-1 no effect | fax-1 no effect | ||
| cfi-1 effect | cfi-1 effect | ||||
| glr-2 (GluR) | unc-3 NO effect | unc-3 NO effect | unc-3 NO effect | unc-3 NO effect | |
| unc-42 NO effect | unc-42 NOeffect | unc-42 NOeffect | |||
| fax-1 NO effect | fax-1 NO effect | fax-1 NO effect | |||
| glr-4 (GluR) | unc-3 NO effect | ||||
| unc-42 effect | |||||
| fax-1 no effect | |||||
| glr-5 (GluR) | unc-3 NO effect | unc-3 NO effect | unc-3 NO effect | unc-3 NO effect | unc-3 NO effect |
| unc-42 effect | unc-42 NOeffect | unc-42 effect | unc-42 effect | ||
| fax-1 no effect | fax-1 NO effect | fax-1 no effect | fax-1 no effect | ||
| opt-3 | unc-3 NO effect | ||||
| unc-42 effect | |||||
| fax-1 effect | |||||
| rig-3 (IgSF) | unc-3 no effect | ||||
| flp-18 (FMRF) | unc-3 no effect |
-
Gray shading: gene normally expressed in this cell. 'Effect' (red) indicate that respective reporter gene fails to be expressed in the respective mutant background in the indicated cells, 'no effect' (green) indicates the opposite.
-
unc- 42, cfi-1 and fax-1 data on non-ACh marker from Wightman et al. (2005) Shaham and Bargmann (2002) and Brockie et al. (2001)
Table 9
Molecular markers for cell identification. The respective markers were crossed with cho-1 or unc-17 fosmid reporters to validate cell identification.
| Neuron | Molecular marker |
|---|---|
| Hermaphrodite | |
| ADFL/R | cat-1::GFP (otIs625) 1 |
| AIA L/R | ttx-3 fosmid::GFP (wgIs68) |
| AIN L/R | ttx-3 fosmid::GFP (wgIs68) |
| AIY L/R | ttx-3 fosmid::GFP (wgIs68) |
| ALN L/R | unc-86 fosmid::YFP (otIs337); lad-2::GFP (otIs439) |
| AS1-11 | unc-3 fosmid::GFP (otIs591) |
| ASJ L/R | DiI/DiO staining |
| AVA L/R | glr-1::DsRed (hdIs30); nmr-1::GFP (akIs3) |
| AVB L/R | acr-15::GFP (wdEx290); sra-11::GFP (otIs123) |
| AVD L/R | glr-1::DsRed (hdIs30); nmr-1::GFP (akIs3) |
| AVE L/R | glr-1::DsRed (hdIs30); nmr-1::GFP (akIs3) |
| AVG | odr-2::DsRed (otEx4452); unc-6 fosmid::GFP (otEx6370) |
| AWB L/R | DiI/DiO staining |
| DA1-9 | unc-3 fosmid::GFP (otIs591); acr-2::GFP (juIs14) |
| DB1-7 | unc-3 fosmid::GFP (otIs591); acr-2::GFP (juIs14) |
| DVA | ser-2::GFP (otIs358) |
| HSN L/R | unc-86 fosmid::YFP (otIs337) |
| IL2 D/V L/R (x6) | unc-86 fosmid::YFP (otIs337) |
| PDA | unc-3 fosmid::GFP (otIs591); ace-3/4::GFP (fpIs1) |
| PDB | unc-3 fosmid::GFP (otIs591) |
| PLN L/R | unc-86 fosmid::YFP (otIs337); lad-2::GFP (otIs439) |
| PVC L/R | nmr-1::GFP (akIs3) |
| PVN L/R | 2 |
| PVP L/R | lin-11 fosmid::GFP (wgIs62); unc-30 fosmid::GFP (wgIs395) |
| RIB L/R | 2 |
| RIF L/R | odr-2::DsRed (otEx4452); unc-6 fosmid::GFP (otEx6370) |
| RIH | cat-1::GFP (otIs625) |
| RIR8 | unc-86 fosmid::YFP (otIs337) |
| RIV L/R | unc-42 fosmid::GFP (wgIs173); lad-2::GFP (otIs439) |
| RMD D/V L/R (x6) | glr-1::DsRed (hdIs30) |
| RMF L/R | 2 |
| RMH L/R | 2 |
| SAA D/V L/R (x4) | lim-4::GFP (mgIs19); lad-2::GFP (otIs439) |
| SAB D V L/R (x3) | unc-4::GFP (vsIs45) |
| SDQ L/R | unc-86 fosmid::YFP (otIs337); lad-2::GFP (otIs439) |
| SIA D/V L/R (x4) | ceh-24::GFP (ccIs4595) |
| SIB D/V L/R (x4) | ceh-24::GFP (ccIs4595) |
| SMB D/V L/R (x4) | lim-4::GFP (mgIs19); lad-2::GFP (otIs439) |
| SMD D/V L/R (x4) | lad-2::GFP (otIs439) |
| URA D/V L/R (x4) | unc-86 fosmid::YFP (otIs337) |
| URB L/R | unc-86 fosmid::YFP (otIs337) |
| URX L/R | flp-10::GFP (otIs92); unc-86 fosmid::YFP (otIs337) |
| VA1-12 | unc-3 fosmid::GFP (otIs591); acr-2::GFP (juIs14) |
| VB1-11 | unc-3 fosmid::GFP (otIs591); acr-2::GFP (juIs14) |
| VC1-6 | lin-11::GFP (nIs106); ida-1::GFP (inIs179) |
| Pharyngeal | |
| I1 L/R | 3 |
| I3 (L) | 3 |
| M1 (R) | 3 |
| M2 L/R | 3 |
| M4 (L) | 3 |
| M5 (L) | 3 |
| MC L/R | 3 |
| M | |
| CEM D/V L/R (x4) | pkd-2::GFP (bxIs14) |
| CA1-9 | ida-1 (inIs179) |
| DVE, DVF | 2 |
| HOB | ida-1 (inIs179) |
| PCB, PCC, SPC | 4 |
| PDC, PGA | 2 |
| PVV | 2 |
| PVX, PVY | 2 |
| PVZ | ida-1 (inIs179) |
| R1A, R2A, R3A, R4A, R6A | 2 |
| SPV | 2 |
-
1Excluded AWA due to lack of overlap of cho-1 fosmid reporter with odr-10::gfp and unc-17 fosmid reporter gfp reporter with gpa-4::mCherry. See Figure 1—figure supplement 1.
-
2Identified based on position and axonal projections because of the lack of available markers.
-
3Pharyngeal neurons identified based on axonal projections which are visible with the unc-17 fosmid reporter.
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
A cellular and regulatory map of the cholinergic nervous system of C. elegans
eLife 4:e12432.
https://doi.org/10.7554/eLife.12432
