Sustained expression of unc-4 homeobox gene and unc-37/Groucho in postmitotic neurons specifies the spatial organization of the cholinergic synapses in C. elegans
- Department of Zoology, University of British Columbia, Canada
- Graduate Program in Cell and Developmental Biology, University of British Columbia, Canada
- Life Sciences Institute, University of British Columbia, Canada
- Djavad Mowafaghian Centre for Brain Health, University of British Columbia, Canada
Figures
Figure 1 with 4 supplements
unc-4 and unc-37 are required for tiled synaptic innervation of DA8 and DA9 neurons.
(A) Schematic of the tiled innervation between DA8 and DA9 neurons of wild-type (top) and synaptic tiling mutants (bottom). (B–D) Representative images of synaptic tiling in wildtype (B), unc-4(e120)…
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Figure 1—source data 1
Quantification of overlap between DA8 and DA9 synaptic domains.
- https://cdn.elifesciences.org/articles/66011/elife-66011-fig1-data1-v1.xlsx
Figure 1—figure supplement 1
Colocalization of GFP::RAB-3 and mCherry::RAB-3 in the DA9 neuron with two synaptic tiling markers.
(A) Representative image of the previously used DA8 and DA9 synaptic marker (wyIs446) in wild-type animals (left). (B) Representative image of the unc-4(e120); wyIs446 animals (left). (C) …
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Figure 1—figure supplement 1—source data 1
Fluorescence intensity of GFP and mCherry channels.
- https://cdn.elifesciences.org/articles/66011/elife-66011-fig1-figsupp1-data1-v1.xlsx
Figure 1—figure supplement 2
Pmig-13::mCherry::rab-3 is not ectopically expressed in DA8 of unc-4(e120) mutants.
(A) Representative image of mizIs3 animals co-expressing his-24::GFPnovo2 in the DA neurons. (B) Representative image of unc-4(e120); mizIs3 co-expressing his-24::GFPnovo2 in the DA neurons. GFP, …
Figure 1—figure supplement 3
unc-4 and unc-37 may function in the same genetic pathway as plx-1.
(A–C) Representative images of synaptic tiling in plx-1(nc36) (A), unc-4(e120);plx-1(nc36) (B), unc-37(e262);plx-1(nc36) (C) mutants. The overlap between the DA8 and DA9 synaptic domains are highligh…
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Figure 1—figure supplement 3—source data 1
Quantification of overlap between DA8 and DA9 synaptic domains in plx-1 mutant animals.
- https://cdn.elifesciences.org/articles/66011/elife-66011-fig1-figsupp3-data1-v1.xlsx
Figure 1—figure supplement 4
plx-1 and rap-2 expression is largely unaffected in unc-4 and unc-37 mutants.
(A–C) Representative image of Pplx-1::GFP in wild-type (A), unc-4(e120) (B), and unc-37(e262) (C) mutants with HIS-24::mCherry in DA8 and DA9. (D) Quantification of animals (n = 100) that expressed …
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Figure 1—figure supplement 4—source data 1
Quantification of animals that expressed Pplx-1::GFP in the anterior and posterior DA8/DA9 of wild-type, unc-4(e120), and unc-37(e262) animals.
- https://cdn.elifesciences.org/articles/66011/elife-66011-fig1-figsupp4-data1-v1.xlsx
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Figure 1—figure supplement 4—source data 2
Quantification of animals (n=100) that expressed Prap-2::GFP in the anterior and posterior DA8/9 of wild-type, unc-4, and unc-37 animals.
- https://cdn.elifesciences.org/articles/66011/elife-66011-fig1-figsupp4-data2-v1.xlsx
Figure 2 with 2 supplements
unc-4 and unc-37 are necessary and sufficient for the tiled innervation of DA8 and DA9 neurons.
(A–D) Representative images of synaptic tiling in unc-4(miz40); mizSi3 (− auxin) (A), unc-4(miz40); mizSi3 (+ auxin) (B), unc-37(miz36); mizSi3 (− auxin) (C), and unc-37(miz36); mizSi3 (+auxin) (D). …
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Figure 2—source data 1
Quantification of overlap of DA8 and DA9 synaptic domains of unc-4(miz40); mizSi3 and unc-37(miz36); mizSi3 animals treated with (+) and without (-) auxin.
- https://cdn.elifesciences.org/articles/66011/elife-66011-fig2-data1-v1.xlsx
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Figure 2—source data 2
Cell specific rescue of unc-4(e120) mutants with unc-4 cDNA using DA-specific promoter (Punc-4c).
- https://cdn.elifesciences.org/articles/66011/elife-66011-fig2-data2-v1.xlsx
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Figure 2—source data 3
Cell specific rescue of unc-37(e262) mutants with unc-37 cDNA using DA-specific promoter (Punc-4c).
- https://cdn.elifesciences.org/articles/66011/elife-66011-fig2-data3-v1.xlsx
Figure 2—figure supplement 1
Genomic structure of the AID strains.
(A) Genomic structure of oxTi365 locus in mizSi3[Punc-4c::TIR1] strains. (B) Genomic structure of unc-4 (top) and unc-37 (bottom) in unc-4(miz40[unc-4::AID::BFP]) and unc-37(miz36[unc-37::AID::BFP]) …
Figure 2—figure supplement 2
DA-neuron-specific degradation of UNC-4 and UNC-37 using AID system.
(A–B) Representative images of unc-4(miz40) in M9 control (A) and auxin (B) treated animals. (C–D) Representative images of unc-37(miz36) in M9 control (C) and auxin (D) treated animals. Magnified …
Figure 3
unc-4 functions in postmitotic DA neurons for the synaptic tiling of DA8 and DA9 neurons.
(A) Experimental design of the temperature shift assay. (B–E) Representative images of synaptic tiling in unc-4(e2322ts) at 16°C (B), unc-4(e2322ts) at 25°C (C), unc-4(e2322ts) embryonic knockdown (D…
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Figure 3—source data 1
Quantification of overlap of DA8 and DA9 synaptic domains.
- https://cdn.elifesciences.org/articles/66011/elife-66011-fig3-data1-v1.xlsx
Figure 4
unc-37 but not unc-4 is required during DA neurogenesis for proper synaptic tiling of DA8 and DA9 neurons.
(A) Experimental design of the embryonic degradation of UNC-4 and UNC-37 using the AID system. (B, C) Representative images of synaptic tiling at L4 stage after embryonic degradation in unc-4(miz40);…
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Figure 4—source data 1
Quantification of overlap of DA8 and DA9 synaptic domains at the L4 stage after embryonic auxin treatment.
- https://cdn.elifesciences.org/articles/66011/elife-66011-fig4-data1-v1.xlsx
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Figure 4—source data 2
Quantification of overlap of DA8 and DA9 synaptic domains at the L2 stage after embryonic auxin treatment.
- https://cdn.elifesciences.org/articles/66011/elife-66011-fig4-data2-v1.xlsx
Figure 5
unc-4 and unc-37 are required in the postmitotic DA neurons to maintain synaptic tiling.
(A) Experimental design of the postembryonic degradation of UNC-4 and UNC-37 using the AID system. (B, C) Representative images of synaptic tiling after early postembryonic degradation in unc-4(miz40…
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Figure 5—source data 1
Quantification of overlap of DA8 and DA9 synaptic domains after postembryonic auxin treatment.
- https://cdn.elifesciences.org/articles/66011/elife-66011-fig5-data1-v1.xlsx
Figure 6 with 1 supplement
unc-4 and unc-37 inhibit canonical Wnt signaling to regulate synaptic tiling.
(A, B) Representative image of synaptic tiling in ectopic expression of ceh-12 under the unc-4c promoter (A) and unc-55A under the unc-4c promoter (B). The overlap between the DA8 and DA9 synaptic …
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Figure 6—source data 1
Quantification of overlap between DA8 and DA9 synaptic domains.
- https://cdn.elifesciences.org/articles/66011/elife-66011-fig6-data1-v1.xlsx
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Figure 6—source data 2
Quantification of overlap between DA8 and DA9 synaptic domains in ceh-12 mutant animals.
- https://cdn.elifesciences.org/articles/66011/elife-66011-fig6-data2-v1.xlsx
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Figure 6—source data 3
Quantification of overlap between DA8 and DA9 synaptic domains in bar-1 mutant animals.
- https://cdn.elifesciences.org/articles/66011/elife-66011-fig6-data3-v1.xlsx
Figure 6—figure supplement 1
ceh-12 expression is derepressed in DA8/9 of unc-4(e120) and unc-37(e262) mutants.
(A–C) Representative images of Pceh-12::GFP with Punc-4c::his-24::mCherry in wild-type (A), unc-4(e120) (B) and unc-37(e262) (C) at the L1 (A–C), L2 (A’–C’), and L4 (A”–C”) stages. Dotted boxes …
Figure 7
Model of UNC-4 and UNC-37 functions in synaptic tiling.
UNC-4 functions with UNC-37 in postmitotic DA neurons to regulate synaptic tiling by inhibiting canonical Wnt signaling. Ectopic expression of CEH-12 and other unknown molecules in DA neurons …
Author response image 1
Author response image 2
Tables
Table 1
Expression of cell fate markers in the anterior or posterior DA8/DA9 neurons.
| DA marker | DB markers | DA9 markers | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| Punc-53 | Punc-129DB | Pacr-16 | Pacr-5 | Pmig-13 | Pglr-4 | Pitr-1 | |||||
| Genotype | Anterior | Posterior | Anterior | Posterior | Anterior | Posterior | Anterior | Posterior | DA9 | DA9 | DA9 |
| Wild type | 94/100 | 94/100 | 2/100 | 4/100 | 0/100 | 0/100 | 0/100 | 0/100 | 100/100 | 100/100 | 100/100 |
| unc-4(e120) | 94/100 | 91/100 | 3/100 | 4/100 | 3/100 | 8/100 | 4/100 | 8/100 | 100/100 | 99/100 | n.d. |
| unc-37(e262) | 92/100 | 92/100 | 1/100 | 8/100 | 2/100 | 24/100 | 1/100 | 30/100 | 100/100 | 93/100 | 98/100 |
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (Caenorhabditis elegans) | unc-4 | WormBase | F26C11.2 | |
| Gene (Caenorhabditis elegans) | unc-37 | WormBase | W02D3.9 | |
| Strain, strain background (C. elegans) | unc-4(e120) | C. elegans stock center (CGC) | CB120 | |
| Strain, strain background (C. elegans) | unc-4(e2322) | C. elegans stock center (CGC) | NC37 | |
| Strain, strain background (C. elegans) | unc-37(e262) | C. elegans stock center (CGC) | CB262 | |
| Strain, strain background (C. elegans) | ceh-12(gk391) | C. elegans stock center (CGC) | VC995 | |
| Strain, strain background (C. elegans) | bar-1(ga80) | C. elegans stock center (CGC) | EW15 | |
| Strain, strain background (C. elegans) | unc-4(miz40) | This study | UJ1017 | unc-4::AID::BFP |
| Strain, strain background (C. elegans) | unc-37(miz36) | This study | UJ1013 | unc-37::AID::BFP |
| Strain, strain background (C. elegans) | mizIs3 | This study | UJ124 | DA8/DA9 synaptic marker |
| Strain, strain background (C. elegans) | mizSi3 | This study | UJ1133 | Punc-4c::TIR1 |
