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. 2017 Jan 4;93(1):80-98.
doi: 10.1016/j.neuron.2016.11.036.

Diversification of C. Elegans Motor Neuron Identity via Selective Effector Gene Repression

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Free PMC article

Diversification of C. Elegans Motor Neuron Identity via Selective Effector Gene Repression

Sze Yen Kerk et al. Neuron. .
Free PMC article

Abstract

A common organizational feature of nervous systems is the existence of groups of neurons that share common traits but can be divided into individual subtypes based on anatomical or molecular features. We elucidate the mechanistic basis of neuronal diversification processes in the context of C.elegans ventral cord motor neurons that share common traits that are directly activated by the terminal selector UNC-3. Diversification of motor neurons into different classes, each characterized by unique patterns of effector gene expression, is controlled by distinct combinations of phylogenetically conserved, class-specific transcriptional repressors. These repressors are continuously required in postmitotic neurons to prevent UNC-3, which is active in all neuron classes, from activating class-specific effector genes in specific motor neuron subsets via discrete cis-regulatory elements. The strategy of antagonizing the activity of broadly acting terminal selectors of neuron identity in a subtype-specific fashion may constitute a general principle of neuron subtype diversification.

Keywords: C. elegans; combinatorial code; maintenance; motor neuron; neuron diversification; neuron subtype; repressor; selective repression; terminal selector; transcription factor.

Figures

Figure 1
Figure 1. C. elegans ventral nerve cord motor neurons
A: Cholinergic MN classes in C. elegans VNC and their relative positions and morphology. Numbers in brackets represent the number of individual MNs in each class. B: Unique combinations of effector gene expression define each unc-3-expressing MN class (as represented by different colors). As with the shared genes of the ACh pathway, class-specific genes are unc-3-dependent since their expression is lost in unc-3 mutants (bounded by red margin). Color-filled rectangles represent expression in the corresponding class; grey rectangles represent absence of expression; superimposed diagonal stripes indicate dim expression. C: UNC-3 is a terminal selector which coordinately regulates the expression of class-specific as well as shared effector genes in cholinergic MNs. This co-regulation is achieved via phylogenetically conserved binding sites, termed COE motifs, found within the cis-regulatory region of these genes. D: The co-activator versus repressor models of regulation: hypothetical models of how UNC-3 selectively controls the expression of class-specific genes. Black and color-filled circles indicate gene is expressed; grey-filled circles indicate gene is not expressed. Class-specific co-activators (A1, A2) and repressors (R1, R2).
Figure 2
Figure 2. bnc-1 is a class-specific regulator of motor neuron identity
A: unc-129 is expressed in DA/DB MNs (class member neurons within white bounding rectangle is magnified in inset on the right) and is regulated by unc-3. In ot721 and ot763 animals (which do not complement each other), unc-129 is derepressed in VA/VB MNs in an unc-3-dependent manner. This phenotype is rescued by the genomic locus of a novel C. elegans gene, bnc-1 (basonuclin 1). Wildtype (WT); all scale bars = 50 µm. B. Quantification for A; error bars show standard deviation (SD). Unpaired t-tests were performed for all mutants compared to WT; ***p < 0.001; n ≥ 13. C: bnc-1 gene locus. D: BNC-1 protein and its orthologs. E: Endogenous bnc-1 locus tagged with mNG shows remarkably specific expression in VA/VB MNs (except for VB1) in the VNC (see Fig.S4A for additional expression elsewhere). Gut auto-fluorescence (*); stitched together from two images of the same worm; showing anterior half of worm. F: bnc-1 cDNA driven by an unc-3 promoter in DA/DB/VA/VB MNs in bnc-1 mutants rescues the unc-129 derepression phenotype in VA/VB, and is sufficient to ectopically repress unc-129 in DA/DB MNs. WT image repeated from A; red pharyngeal expression marks unc-3prom::bnc-1 transgene. G: Expression of the unc-3-dependent class-specific effector genes unc-53 (DA/AS), unc-8 (DA/DB/AS) and acr-16 (DB) are derepressed in VA/VB MNs of bnc-1 mutants. H: Quantification for G; error bars show SD. Superimposed diagonal stripes indicate dim expression. Unpaired t-tests were performed for all mutants compared to WT; ***p < 0.001; n ≥ 13. I: Genetic model depicting BNC-1 repressing DA/DB-specific effector genes in VA/VB MNs.
Figure 3
Figure 3. mab-9 is a class-specific regulator of motor neuron identity
A: Expression of unc-129 is lost in mab-9(ot720) animals (phenocopied by gk396730 and e2410). In the double mutant, the phenotype of bnc-1 is epistatic to that of mab-9, indicating that MAB-9 represses bnc-1. mab-9 cDNA driven by the unc-3 promoter in mab-9 mutants rescues the unc-129 expression loss phenotype in DA/DB, and is sufficient to ectopically derepress unc-129 in VA/VB MNs. Similarly, unc-53 expression in DA MNs is lost (albeit only in the anterior half of the VNC) in mab-9(ot788). WT images repeated from Fig.2A,G; red pharyngeal expression marks unc-3prom::mab-9 transgene; all scale bars = 50 µm. B: Quantification for A; error bars show SD. Unpaired t-tests were performed for all mutants compared to WT; ***p < 0.001; n ≥ 13. C: mab-9 gene locus. The endogenous reporter is not visible in the VNC. D: bnc-1 is derepressed in DA/DB MNs in mab-9 mutants. WT image repeated from Fig.2E. A bnc-1 promoter 1.8 kb upstream of the start codon recapitulates the VA/VB-specific expression of endogenous bnc-1. Mutation (mut-) of MAB-9 binding sites causes bnc-1 to be derepressed in DA/DB MNs. All rfp represents mchopti+h2b. See Fig.S1H for quantification. E: bnc-1 fluorescent reporters of the endogenous gene locus (CRISPR; see Fig.2E), a fosmid (see Fig.S1G), the rescue construct used in Fig.2A,B (see Fig.S1G), and the abovementioned 1.8 kb promoter (see Fig.3D) express specifically in VA/VB MNs. Deletion of MAB-9 binding sites (white-filled vertical rectangle) causes bnc-1 to be derepressed in DA/DB MNs. See Fig.S1H for quantification. F: del-1 is expressed in VA/VB MNs and is regulated by unc-3. In mab-9 mutants, del-1 is derepressed in DA/DB MNs in an unc-3-dependent manner. The unc-3prom::mab-9 transgene rescues this phenotype, and is sufficient to ectopically repress del-1 in VA/VB MNs. Similarly, unc-3-dependent inx-12 which is VA/VB-specific is also derepressed in DA/DB MNs in mab-9 mutants. G: Quantification for F and the derepression of unc-3-dependent, VA/VB-specific lgc-36 in DA/DB MNs in mab-9 mutants (see Fig.S1I for images); error bars show SD. Unpaired t-tests were performed for all mutants compared to WT; ***p < 0.001; n ≥ 13. H: Genetic model depicting MAB-9 repressing BNC-1 as well as VA/VB-specific effector genes in DA/DB MNs.
Figure 4
Figure 4. Additional class-specific repressors control motor neuron identity
A: In ot718 animals, unc-129 is derepressed in AS MNs (phenocopied by the unc-55 mutant allele, e1170) and this phenotype is unc-3-dependent. ot718 fails to complement e1170 and is thus confirmed to be an unc-55 mutant allele. del-1 expression is unaffected in unc-55 mutants, but is derepressed in AS MNs in mab-9; unc-55 double mutants – indicating a redundant role of these two genes. The individual defects of bnc-1, mab-9, and unc-55 mutants are additive since the expression of unc-129 in bnc-1; unc-55 and del-1 in mab-9; unc-55 double mutants is no longer class-specific. WT and mab-9 mutant images repeated from Fig.2A and Fig.3F, respectively; all images show anterior half of worm; all scale bars = 50 µm. B: unc-55 gene locus. C: DA/DB/VA/VB-specific and unc-3-dependent effector genes acr-2, dbl-1, and slo-2 are derepressed in AS MNs in unc-55 mutants. See Fig.S3A for quantification. D: In unc-4 mutants, acr-16 is derepressed in DA MNs in an unc-3-dependent manner. Additive effect of double mutant bnc-1; unc-4 causes acr-16 to no longer be as class-specific. E: In vab-7 mutants, unc-53 is derepressed in DB MNs in an unc-3-dependent manner. Additive effect of double mutant bnc-1; vab-7 causes unc-53 to no longer be as class-specific. F: Quantification for A, D, and E; error bars show SD. Unpaired t-tests were performed for all mutants compared to WT unless otherwise indicated; ***p < 0.001; n ≥ 13. G: Genetic model depicting the interactions of repressors on class-specific effector genes in VNC MNs. In AS MNs, acr-16 and acr-5 are likely repressed by as yet unidentified AS-specific repressors, perhaps redundantly with UNC-55.
Figure 5
Figure 5. A repressor regulatory logic also operates in head SAB motor neurons
A: SAB MNs in the RVG and their relative positions and morphology. B: unc-129 expression is specific to SABD and is unc-3-dependent. This expression is lost in mab-9 mutants in a bnc-1-dependent manner, indicating that MAB-9 represses bnc-1 which represses unc-129. Similarly, unc-129 expression in SABD is lost in unc-4 mutants. This effect is likely also indirect with UNC-4 possibly repressing an unidentified repressor of unc-129 (note that e26 disrupts UNC-4 interaction with the Groucho-like corepressor UNC-37). This repressor is not bnc-1 as the bnc-1 mutant phenotype is not epistatic to that of unc-4. On the other hand, unc-129 is derepressed in SABVs (albeit incompletely) in unc-4 mutants in a mab-9-dependent manner, indicating that UNC-4 represses mab-9. As in SABD, mab-9 is likely repressing bnc-1 which represses unc-129. This predicts that bnc-1 mutants would phenocopy those of unc-4 – which is not the case. UNC-4 might independently to, and redundantly with bnc-1 be repressing unc-129 in SABVs. Fischer’s exact tests were performed for all mutants compared to WT; ***p < 0.001; n ≥ 25. C: del-1 expression is specific to SABVs and is unc-3-dependent. In mab-9 mutants, del-1 is derepressed in SABD. In unc-4 mutants, del-1 expression is lost in SABVs in a mab-9-dependent manner, indicating that UNC-4 represses mab-9 which represses del-1. Fischer’s exact tests were performed for all mutants compared to WT; ***p < 0.001; n ≥ 20. D: Effect of repressor mutants on class-specific effector genes in SAB MNs. Note expression pattern of repressor genes (see Fig.S4A). Red bounding lines highlight changes in effector gene expression in repressor mutants compared to WT. E: Genetic model depicting the interactions of repressors on class-specific effector genes in SAB MNs. F: glr-4 is expressed in SAB MNs in an unc-3-dependent manner, but expression is largely absent in VNC MNs. In cfi-1(ot786), glr-4 is derepressed in DA/DB MNs (phenocopied by ky651) in an unc-3-dependent manner. This phenotype is rescued by a fosmid containing the cfi-1 locus. Additionally, in lin-13(ot785), glr-4 is derepressed in AS MNs (phenocopied by n770). Error bars show SD; unpaired t-tests were performed for all mutants compared to WT; ***p < 0.001; n ≥ 13. See Fig.S4C,D for images. G: cfi-1 gene locus. H: lin-13 gene locus. Not shown are four regulatory mutations in ot785.
Figure 6
Figure 6. Repressor binding sites adjacent to COE motif mediate class specificity of motor neuron effector gene expression
White-filled vertical rectangles represent mutated sites. Expression observed in corresponding MN class (+); absence of expression (−); although largely absent, weak expression in a small number of MNs occasionally observed (^); unexpected result to be addressed (*). All scale bars = 50 µm. A,B: Mutation of BNC-1 binding site results in unc-129 derepression in VA/VB and AS MNs. *The BNC-1 site harbors two UNC-55 site consensus motifs with one mismatch in each. These two repressors may be sharing the same site in this case. del-1prom::gfp marks VA/VB MNs. C,D: Mutating the COE motif causes acr-16 expression to be lost. When the BNC-1 binding site is mutated, acr-16 becomes derepressed in VA/VB and DA MNs. *The BNC-1 site does not resemble that of UNC-4 nor is there an UNC-4 site near that of BNC-1. This result nonetheless supports the notion that effector genes are repressed at the cis-regulatory level to achieve class specificity. 1.8 kb reporter image repeated from Fig.4D; del-1prom::gfp marks VA/VB MNs. E,F: Mutation of two MAB-9 binding sites adjacent to the COE motif proximal to the start codon results in del-1 derepression in DA/DB MNs. *Expression in anterior VA/VBs (from VA2/VB3 and upward) is at times repressed – hinting at finer subclass regulation. No derepression is observed when all four UNC-55 sites are mutated, but deletion of both validated MAB-9 and all four UNC-55 sites results in del-1 derepression in DA/DB and AS MNs. unc-129prom::gfp marks DA/DB MNs. G,H: Mutating the two CFI-1 binding sites flanking the COE motif causes glr-4 to be derepressed in DA/DB MNs, albeit in a variable manner. This effect is potentiated when all four sites are mutated. I: Quantification for B, D, F, and H; error bars show SD. Superimposed diagonal stripes indicate dim expression. At least three independent transgenic lines were assessed although only the most representative line is shown. Unpaired t-tests were performed comparing each line with its corresponding non-mutated control; ***p < 0.001; *p < 0.05; n ≥ 10.
Figure 7
Figure 7. BNC-1 and MAB-9 are continuously required to postdevelopmentally maintain motor neuron class identity
A: Postdevelopmental UNC-3 degradation by treating young adult, conditional unc-3 worms with auxin for 3 days, results in reduction of unc-129 and del-1 expression. All scale bars = 50 µm. B: Quantification for A; error bars show SD. UNC-3 function is slightly disrupted when fused to AID. Superimposed diagonal stripes indicate dim expression. Unpaired t-tests were performed comparing each auxin-treated condition with its corresponding EtOH control; ***p < 0.001; n ≥ 13. Data of unc-129 and del-1 expression in WT and unc-3 mutants repeated from Fig.2B and Fig.3G, respectively. C: Postdevelopmental BNC-1 degradation by treating conditional bnc-1 worms at the L4 stage (just before entry into adulthood) with auxin for 1 day results in unc-129 derepression in VA/VB MNs. D: First-generation progenies (F1) of conditional bnc-1 worms constitutively grown on auxin phenocopy bnc-1 mutants in terms of unc-129 derepression. When auxin is removed at the L4 stage, repression of unc-129 in VA/VB MNs is restored after 3 days. E: Quantification for C and D; error bars show SD. BNC-1 function is slightly disrupted when fused to AID. Unpaired t-tests were performed comparing each experimental condition with its corresponding untreated control; ***p < 0.001; n ≥ 13. F: AID-tagged BNC-1 is degraded within 1 h upon auxin treatment. Upon auxin removal, BNC-1 expression is restored within 6 hrs. G: Postdevelopmental MAB-9 degradation by treating young adult conditional mab-9 worms with auxin for 2 days results in del-1 derepression in DA MNs. MAB-9 function in DB MNs is disrupted non-conditionally when fused to AID. H: F1 conditional mab-9 worms constitutively grown on auxin phenocopy mab-9 mutants in terms of del-1 derepression. When auxin is removed at the L4 stage, repression of del-1 in DB MNs is not restored even after 4 days, likely due to the hypomorphic nature of this mab-9 allele. I: Quantification for G and H; error bars show SD. Unpaired t-tests were performed comparing each experimental condition with its corresponding untreated control; ***p < 0.001; n ≥ 13.
Figure 8
Figure 8. Proposed general principle of motor neuron diversification
A: Repressor mutant effects on class-specific effector genes in VNC MNs. Only derepression effects are shown within red bounding rectangles. Red crosses indicate absence of a repressor in its mutant. The hypothetical combined effects of all repressor mutants predict the loss of the unique combinations of class-specific effector genes which define MN classes, leading to the loss of MN diversity (compare the first and last tables bounded by blue margins). B: Model depicting the transcriptional activity of a broadly acting terminal selector of neuron identity (A) being counteracted upon by subtype-specific repressors (R1–4) at the target effector gene level (circles represent effector genes – black- and grey-filled circles indicate that the gene, respectively, is and is not expressed; colored rectangles above the circles represent cis-regulatory binding sites of trans-acting factors) to generate unique combinations of effector genes which specify distinct neuron subtypes. We propose that this strategy may constitute a general principle of neuron identity diversification. Our model highlights several important features:

subtype-specific repressors counteract in parallel the transcriptional activity of the broadly acting terminal selector

each subtype possesses a unique combination of repressors to control the unique effector gene profile that defines the subtype identity

repressors work via cognate binding motifs adjacent to the activator motif within the cis-regulatory region of the effector gene

subtype-specific expression of an effector gene is determined by its combination of repressor binding motifs

continuous presence of these trans-acting factors are required to maintain subtype identity throughout the lifetime of the neuron.

See Fig.S6A for a similar model detailed with the incorporation of results from this study.

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