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A cellular and regulatory map of the cholinergic nervous system of C. elegans

  1. Laura Pereira  Is a corresponding author
  2. Paschalis Kratsios
  3. Esther Serrano-Saiz
  4. Hila Sheftel
  5. Avi E Mayo
  6. David H Hall
  7. John G White
  8. Brigitte LeBoeuf
  9. L Rene Garcia
  10. Uri Alon
  11. Oliver Hobert  Is a corresponding author
  1. Columbia University, United States
  2. Howard Hughes Medical Institute, Columbia University, United States
  3. Weizmann Institute of Science, Israel
  4. Albert Einstein College of Medicine, United States
  5. MRC Laboratory of Molecular Biology, United Kingdom
  6. Texas A&M University, United States
  7. Howard Hughes Medical Institute, Texas A&M University, United States
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Cite this article as: eLife 2015;4:e12432 doi: 10.7554/eLife.12432
10 figures, 1 video and 9 tables

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.

https://doi.org/10.7554/eLife.12432.004
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).

https://doi.org/10.7554/eLife.12432.005
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.

https://doi.org/10.7554/eLife.12432.006
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.

https://doi.org/10.7554/eLife.12432.007
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.

https://doi.org/10.7554/eLife.12432.008
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.

https://doi.org/10.7554/eLife.12432.012
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).

https://doi.org/10.7554/eLife.12432.014
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.

https://doi.org/10.7554/eLife.12432.015
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.

https://doi.org/10.7554/eLife.12432.017
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.

https://doi.org/10.7554/eLife.12432.018
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.

https://doi.org/10.7554/eLife.12432.020
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).

https://doi.org/10.7554/eLife.12432.021
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.

https://doi.org/10.7554/eLife.12432.022
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.

https://doi.org/10.7554/eLife.12432.025
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). (Bcho-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.

https://doi.org/10.7554/eLife.12432.026

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.

https://doi.org/10.7554/eLife.12432.011

Tables

Table 1

Cholinergic neurons in the hermaphrodite.

https://doi.org/10.7554/eLife.12432.003
Neuron typeNeuron classVAChT/ChAT1ChT 2AChE 3Co-transmitterPrevious
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

 

 

 

 

 

  1. See the legend to Figure 2A and Table 2 for notes on neuron classification. Data for the male nervous system is shown in Table 5. '+' indicate relative expression levels. See Figure 1 for images.

  2. *Expression of cho-1 in the RIB neurons is strong but unc-17 expression is, at best, very dim.

  3. 1Gray shading indicates unc-17/cha-1 (VAChT/ChAT) expression as assessed by fosmid reporter and antibody staining.

  4. 2Gray shading indicates cho-1 (ChT) expression as assessed by fosmid reporters.

  5. 3Gray shading indicates reporters expression of one of the C. elegans ace (AChE) genes.

  6. 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.

  7. 8Previously proposed to be DVC (Duerr et al., 2008) but based on position and markers reassigned to DVA.

  8. 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.

https://doi.org/10.7554/eLife.12432.009
Neuron classNeuronNeurotransmitterNotes
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.

https://doi.org/10.7554/eLife.12432.010
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.

https://doi.org/10.7554/eLife.12432.013
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                  
  1. Yellow: Glu, red: ACh, green: Aminergic, blue: GABA.

  2. opp.: command interneurons control opposite drives (forward/reverse).

  3. 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.

https://doi.org/10.7554/eLife.12432.016
Neuron
type
Neuron classunc-17 fosmid
expression
cho-1 fosmid
expression
Co-transmitterPrevious 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
  1. *CA7-9 do not express cho-1 and have lower levels of unc-17 than CA1-6.

  2. 6Our unpublished data.

  3. 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.

https://doi.org/10.7554/eLife.12432.019
Gene*DNA binding domainNeuron 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 yesyesyes 
PDB  yesyesn.d.
DVA yesyesno
PVC yesyesno
AVA yes yesno
AVB yesyesno
AVD yesyesno
AVE yesyesno
PVN yesn.a. n.d.
unc-42
(Prd-type)
Homeodomain RIV yesyes n.d. Glu(ASH) 7
RMD yesyes yes
SMD yesyesn.d.
SIB yes yesn.d.
AVA no no yes1
AVD no no yes1
AVE no no yes1
lim-4(Lhx6/8) Homeodomain AWB yesyesyes2 
SMB yesyesyes3
RIV nonon.d.
lin-11 (Lhx1) Homeodomain ADF nonono Glu (ASG, ADL) 7
PVP yes yes yes4
unc-30
(Pitx)
Homeodomain PVP yes yes yes4GABA (DD, VD) 9
RIH no no n.d.
unc-86
(Brn3)
Homeodomain CEM (male) yes yes yes5Glu (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
  1. *Vertebrate orthologs in parenthesis. All neuron classes listed express the respective transcription factor tested.

  2. **'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.

https://doi.org/10.7554/eLife.12432.023
  unc-3 (+) neuronsCholinergic 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
INTERCommand 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 affected   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    
  1. 2Cho-1 not expressed in these neurons.

  2. 3As previously reported by Kratsios et al. (2011), (2015).

Table 8

Transcription factors affecting command interneuron differentiation.

https://doi.org/10.7554/eLife.12432.024

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        
  1. 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.

  2. 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.

https://doi.org/10.7554/eLife.12432.027
NeuronMolecular 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
  1. 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.

  2. 2Identified based on position and axonal projections because of the lack of available markers.

  3. 3Pharyngeal neurons identified based on axonal projections which are visible with the unc-17 fosmid reporter.

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