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Temporal transitions in the post-mitotic nervous system of Caenorhabditis elegans

Nature volume 600, pages 93–99 (2021)Cite this article

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Abstract

In most animals, the majority of the nervous system is generated and assembled into neuronal circuits during embryonic development1. However, during juvenile stages, nervous systems still undergo extensive anatomical and functional changes to eventually form a fully mature nervous system by the adult stage2,3. The molecular changes in post-mitotic neurons across post-embryonic development and the genetic programs that control these temporal transitions are not well understood4,5. Here, using the model system Caenorhabditis elegans, we comprehensively characterized the distinct functional states (locomotor behaviour) and the corresponding distinct molecular states (transcriptome) of the post-mitotic nervous system across temporal transitions during post-embryonic development. We observed pervasive, neuron-type-specific changes in gene expression, many of which are controlled by the developmental upregulation of the conserved heterochronic microRNA LIN-4 and the subsequent promotion of a mature neuronal transcriptional program through the repression of its target, the transcription factor lin-14. The functional relevance of these molecular transitions are exemplified by a temporally regulated target gene of the LIN-14 transcription factor, nlp-45, a neuropeptide-encoding gene, which we find is required for several distinct temporal transitions in exploratory activity during post-embryonic development. Our study provides insights into regulatory strategies that control neuron-type-specific gene batteries to modulate distinct behavioural states across temporal, sexual and environmental dimensions of post-embryonic development.

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Fig. 1: Temporal transitions in locomotor behaviour and the neuronal transcriptome.
Fig. 2: lin-4 and lin-14 control temporal transitions in exploratory behaviour and neuronal transcriptome.
Fig. 3: lin-4 and lin-14 control developmental changes in exploratory behaviour through regulation of nlp-45.
Fig. 4: Mechanism of nlp-45 gene expression regulation across spatial, temporal, sexual and environmental dimensions of post-embryonic development.

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Data availability

Raw and processed RNA-seq data are available under Gene Expression Omnibus (GEO) accession GSE158274. Raw and processed ChIP–seq data are available under GEO accession GSE181288. Raw microscope images of gene expression reporters with NeuroPAL for neuronal cell ID are available at https://zenodo.org/communities/neuropal.

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Acknowledgements

We thank Q. Chen and A. Bhattacharya for generating transgenic lines; D. Rahe for help with INTACT optimization; A. Romero and E. Yemini for help with worm tracking; L. Cochella, M. P. Hart, I. Beets and members of the Hobert laboratory for comments on the manuscript; and staff at Wormbase and the CGC for providing resources and reagents. This work was funded by the NIH K99 HD098371, National Research Council of Canada (Holmes Award) and by the Howard Hughes Medical Institute. Some strains were provided by the CGC, which is funded by the NIH Office of Research Infrastructure Program (P40 OLD010440).

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  1. HaoSheng Sun

    Present address: Department of Cell, Developmental and Integrative Biology, Heersink School of Medicine, University of Alabama at Birmingham, Birmingham, AL, USA

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  1. Department of Biological Sciences, Howard Hughes Medical Institute, Columbia University, New York, NY, USA

    HaoSheng Sun & Oliver Hobert

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  2. Oliver Hobert
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Contributions

Conceptualization: H.S. and O.H. Methodology: H.S. Validation: H.S. Formal analysis: H.S. Investigation: H.S. Resources: H.S. Writing—original draft: H.S. Writing—review and editing: H.S. and O.H. Visualization: H.S. Supervision: H.S. and O.H. Funding acquisition: H.S. and O.H.

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Correspondence to HaoSheng Sun or Oliver Hobert.

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Peer review information Nature thanks Douglas Portman and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data figures and tables

Extended Data Fig. 1 Developmental transitions in neuronal transcriptome across post-embryonic life stages.

a, Schematic and experimental design for INTACT sample collection, protocol, and data analysis for neuronal transcriptome profiling across development. Representative images of the pan-neuronal INTACT strain as well as neuronal nuclei after immunoprecipitation (IP) are shown in bottom left panels. Representative tracks from IGV are shown for input and neuronal IP samples to demonstrate IP enrichment for pan-neuronally expressed gene, rab-3. b–d, Principal component analysis (PCA) of neuronal transcriptome across post-embryonic development was conducted using DESeq2 in R studio43. Both batch as well as developmental stage were taken as factors for analysis. Each dot represents a replicate in the RNA-seq analysis. b, PC2 vs PC1. PC1 and 2 delineated the transitions between early larval (L1 and L2) stages and late larval (L4)/adult stages, and between all larval (L1 through L4) stages and the adult stage, respectively. c, PC3 vs PC1. PC3 largely accounted for variation as a result of batch. d, PC4 vs PC1. PC4 largely accounted for L2 specific changes. e, The numbers of significant (Padj<0.01) increases/decreases in gene expression are shown for each stage transition. f, Venn diagram of developmental changes in neuronal gene expression across different stage transitions, showing some overlaps but also distinct developmental changes across each stage transition. g, Gene ontology analysis of the 2639 developmentally regulated genes using the Enrichment Tool from Wormbase. h, Top: heat map of the 249 developmentally regulated genes between L1 and L2 stages across post-embryonic development. In addition to developmentally upregulated and downregulated genes, there was a small subset of genes that showed specific upregulation at the L2 stage. Bottom: gene ontology analysis of these genes using the Enrichment Tool from Wormbase. i, Top: heat map of the 448 developmentally regulated genes between L2 and L4 stages across post-embryonic development. Bottom: gene ontology analysis of these genes using the Enrichment Tool from Wormbase. j, Top: heat map of the 510 developmentally regulated genes between L4 and adult stages across post-embryonic development. Bottom: gene ontology analysis of these genes using the Enrichment Tool from Wormbase.

Extended Data Fig. 2 Temporal transitions in nervous system gene expression across C. elegans post-embryonic development.

For all panels, validations of developmentally regulated genes with expression reporters are shown. On top are the scattered dot plots (each point represents a single replicate, n in bracket) of the normalized read counts across all developmental stages from the neuronal INTACT/RNA-seq profiling. Mean +/- SEM are shown for each stage. Adjusted P values (Padj), as calculated by DESeq2, for each developmental comparison are below. Below the RNA-seq read count plots are the schematics and allele names of the expression reporters. Below that are representative confocal microscopy images of the expression reporters across development. Specific regions/neurons are labelled with dotted lines: those labelled with black dotted lines/names are not altered developmentally while those labelled with green and red lines/names demonstrate, respectively, decreases and increases in expression across development. Those labelled with brown lines/names demonstrate both increases and decreases in expression in the same neurons across development. Red scale bars (10µm) are on the bottom right of all representative images. For all panels, L1 through L4 represent the first through the fourth larval stage animals. For a, additional quantification of fluorescence intensity is also shown at the bottom. Two-sided t-test with post hoc correction P values and n (in bracket) are shown. Additional details are included in Supplementary Table 6. a, Metabotropic glutamate receptor gbb-2, as validated with a translational fosmid reporter (gfp), shows expression in the same set of neurons across development31, although the intensity of expression is decreased across development, including that in the nerve ring as measured with fluorescence intensity. b, Gap junction molecule inx-19, as validated with a transcriptional fosmid reporter (sl2::yfp::h2b), loses expression in sixteen neuronal classes across development and gains expression in the AVA neuron upon entry into adulthood. c, Transcription cofactor mab-10, as validated with an endogenous translational reporter (3xflag::mcherry) engineered with CRISPR/Cas9, gains expression across the nervous system amongst other tissue during transition into the L4 stage that is further upregulated in adulthood. The RNA prediction matches well with previous RNA FISH analysis53. The difference between RNA data and protein reporter expression is consistent with previous characterized post-transcriptional regulation by LIN-4154. d, Nuclear hormone receptor daf-12, as validated with an endogenous translational reporter (gfp) engineered with CRISPR-Cas9, shows increased expression broadly across the nervous system during early/mid-larval stage and then decreased expression upon transition into late larval/adult stage. e, Homeodoman transcription factor tab-1, as validated with an endogenous translational reporter (gfp) engineered with CRISPR-Cas9, loses expression in five classes of neuron during early larval development. f, Immunoglobulin-like domain molecule oig-8, as validated with a translational fosmid reporter (gfp), loses expression in two classes of neuron during early larval development.

Extended Data Fig. 3 Temporal transitions in nervous system gene expression across C. elegans post-embryonic development for the neuropeptide family.

a, Heat map of all neuronally enriched neuropeptides across post-embryonic development. Values were z-score normalized and plotted using pheatmap in R studio. Each row represents a single gene, and each column represents a single RNA-seq replicate. For b–k, validations of developmentally regulated genes with expression reporters are shown. On top (left for b) are the scattered dot plots (each point represents a single replicate, n in bracket) of the normalized read counts across all developmental stages from the neuronal INTACT/RNA-seq profiling. Mean +/- SEM are shown for each stage. Adjusted P values (Padj), as calculated by DESeq2, for each developmental comparison are below. Below the RNA-seq read count plots are the schematics and allele names of the expression reporters. Below that (to the right for b, g) are representative confocal microscopy images of the expression reporters across development. Specific regions/neurons are labelled with dotted lines: those labelled with black dotted lines/names are not altered developmentally while those labelled with green and red lines/names demonstrate, respectively, decreases and increases in expression across development. Those labelled with brown lines/names demonstrate both increases and decreases in expression in the same neurons across development. d and v in brackets denotes dim and variable expression, respectively. Red scale bars (10µm) are on the bottom right of all representative images. For all panels, L1 through L4 represent the first through the fourth larval stage animals. Additional details are included in Supplementary Table 6. b, Other than some remnant expression from embryo in early L1 animals, nlp-45 gains expression progressively in a number of neurons across development. c, Neuropeptide-encoding gene ins-6, as validated with an endogenous reporter (t2a::3xnls::gfp) engineered with CRISPR-Cas9, gains expression in ASJ across the L2->L3 transition. Variable AWA expression (not shown) is detected in L3 animals onwards. d, Neuropeptide-encoding gene ins-9, as validated with an endogenous reporter (t2a::3xnls::gfp) engineered with CRISPR-Cas9, gains expression in a number of neurons as it enters L3/L4 stages and loses expression in a subset of these neurons upon entry into adulthood. Extremely variable and dim RVG neuron expression (VB1/2, not shown) is detected in L3/L4 animals. e, Neuropeptide-encoding gene flp-26, as validated with an endogenous reporter (t2a::3xnls::gfp) engineered with CRISPR-Cas9, loses expression in M4 and I6 pharyngeal neurons across early larval development and loses expression in PVQ as it enters late larval/adult stages. f, Neuropeptide-encoding gene flp-28, as validated with an endogenous reporter (t2a::3xnls::gfp) engineered with CRISPR-Cas9, gains expression in hermaphrodite specific neurons (VC, HSN) as it enters late larval/adult stages. Head/tail neurons do not appear to be developmentally regulated in this reporter (images not shown). g, Neuropeptide-encoding gene nlp-50, as validated with an endogenous reporter (t2a::3xnls::gfp) engineered with CRISPR-Cas9, gains expression in a number of neurons across development. It also loses expression in the RID neuron during early larval development. Additional quantification of RIP and BAG fluorescence intensity across development is also shown at the bottom. Two-sided t-test with post hoc correction P values and n in brackets are shown. h, Neuropeptide-encoding gene flp-17, as validated with a promoter fusion reporter (gfp), loses and gains expression in nine and two classes of neurons, respectively, across post-embryonic development. i, Neuropeptide-encoding gene flp-14, as validated with an endogenous reporter (t2a::3xnls::gfp) engineered with CRISPR-Cas9, loses and gains expression in one (LUA) and two (AVB and AVG) classes of neurons as it enters late larval/adult stages, respectively. j, Neuropeptide-encoding gene nlp-13, as validated with an endogenous reporter (t2a::3xnls::gfp) engineered with CRISPR-Cas9, gains expression in the ventral nerve cord neurons (DA, VA, VD, VC) across development. Head/tail neurons do not appear to be developmentally regulated in this reporter (images not shown). k, Neuropeptide receptor gene npr-17, as validated with an endogenous translational reporter (gfp) engineered with CRISPR-Cas9, shows decreased and increased expression in four and one classes of neurons, respectively.

Extended Data Fig. 4 Temporal transitions in nervous system gene expression across C. elegans post-embryonic development for the receptor-type guanylyl cyclases (rGCs) family.

a, Heat map of all neuronally enriched receptor-type guanylyl cyclases (rGCs) across post-embryonic development. Values were z-score normalized and plotted using pheatmap in R studio. Each row represents a single gene, and each column represents a single RNA-seq replicate. For b–d, validations of developmentally regulated genes with expression reporters are shown. On top are the scattered dot plots (each point represents a single replicate, n in bracket) of the normalized read counts across all developmental stages from the neuronal INTACT/RNA-seq profiling. Mean +/- SEM are shown for each stage. Adjusted P values (Padj), as calculated by DESeq2, for each developmental comparison are below. Below the RNA-seq read count plots are the schematics and allele names of the expression reporters. Below that are representative confocal microscopy images of the expression reporters across development. Specific regions/neurons labelled with dotted lines: those labelled with black dotted lines/names are not altered developmentally while those labelled with green and red lines/names demonstrate, respectively, decreases and increases in expression across development. Red scale bars (10µm) are on the bottom right of all representative images. For all panels, L1 through L4 represent the first through the fourth larval stage animals. Additional details are included in Supplementary Table 6. b, rGC gcy-5, as validated with a transcriptional fosmid reporter (sl2::1xnls::gfp), shows decreased expression in ASER and increased expression in RIG, as measured with fluorescence intensity, across development. Quantifications of fluorescence intensity are shown at the bottom. Two-sided t-test with post hoc correction P values and n in brackets are shown. c, rGC gcy-21, as validated with a promoter fusion reporter (gfp), loses expression in three neuronal classes across the L1->L2 transition. d, rGC gcy-12, as validated with a promoter fusion reporter (gfp), gains expression in A and B type motor neurons across mid/late larval development.

Extended Data Fig. 5 Expression of lin-14 is downregulated in the nervous system amongst other tissues across post-embryonic development in a lin-4 dependent manner in hermaphrodite animals.

a, Expression of lin-4 is turned on in the nervous system amongst other tissues during the L1->L2 transition. Schematic of the lin-4 fosmid expression reagent, in which the lin-4 pre-miRNA sequence was replaced with YFP, is shown on the left. Representative images of lin-4 expression in L1 and L2 animals are shown on the right. Ellipse and polygon outline the anterior and lateral/ventral neuronal ganglions respectively. b, Expression of lin-14 is downregulated in the nervous system amongst other tissues across post-embryonic development in a lin-4 dependent manner in hermaphrodite animals. Schematic of the lin-14 translational GFP allele, as engineered by CRISPR-Cas9, as well as lin-14 gain-of-function (gf) alleles (ot1087/ot1149/ot1150/ot1151), where a 466bp region containing all seven lin-4 repressive binding sites is deleted, are shown on the upper left. All 4 gf alleles represent the same molecular lesion but resulted from independent CRISPR-Cas9 mediated deletions. Quantification of LIN-14::GFP expression in the lateral/ventral ganglion is shown on the upper right. Two-sided t-test with post hoc correction P values and n (in bracket) are shown. On the bottom are the representative images of the lin-14::GFP allele across post-embryonic development in control, lin-4(e912)-null, and lin-14(ot1087) gf animals. Ellipse and polygon outline the anterior and lateral/ventral neuronal ganglions respectively. Red scale bars (10µm) are on the bottom right of all representative images. Expression of lin-14 is still detectable in the adult hermaphrodite. lin-14 expression is upregulated in lin-4(e912) null and lin-14(ot1087) gf animals across development. The incomplete juvenization of lin-14 expression across development in the lin-4 null mutant suggests additional mechanisms beyond lin-4 that downregulate lin-14 across post-embryonic development.

Extended Data Fig. 6 lin-4 controls a subset of the developmentally regulated gene battery through direct repression of lin-14, and not lin-28.

a, lin-4(e912) null mutation juvenizes a subset of the adult control(Ctl) neuronal transcriptome to resemble that of the L1 Ctl neuronal transcriptome through direct de-repression of lin-14 and not lin-28. Principal component analysis (PCA) of the neuronal transcriptomes across post-embryonic development and across genotypes was conducted using DESeq2 in R studio. Each dot represents a replicate in the RNA-seq analysis. b, Correlation between developmentally gene expression changes (log2FoldChange[Adult Expression/L1 Expression]) with gene expression changes in lin-4(e912) null mutation (log2FoldChange[Adult lin-4(e912) null expression /Adult control expression], left), in lin-14(ot1149) gain-of-function (gf) mutation (log2FoldChange[Adult lin-14(ot1149) gf expression/Adult control expression], middle), and in lin-28(ot1154) gf mutation (log2FoldChange[Adult lin-28(ot1154) gf expression/Adult control expression], right). Linear regression was fitted through each set of data points, and the equation and R2 values are shown for each. lin-4 null and lin-14 gf mutations accounted for some of the developmentally gene expression changes between L1 and adult, while lin-28 gf mutation did not. c, Correlation between gene expression changes in lin-4(e912) null mutation (log2FoldChange[Adult lin-4(e912) null expression /Adult control expression]) with gene expression changes in lin-14(ot1149) gf mutation (log2FoldChange[Adult lin-14(ot1149) gf expression/Adult control expression], left), and in lin-28(ot1154) gf mutation (log2FoldChange[Adult lin-28(ot1154) gf expression/Adult control expression], right). Linear regression was fitted through each set of data points, and the equation and R2 values are shown for each. lin-14 gain of function mutation accounted for most of the changes observed in the lin-4 null mutation, but lin-28 gain of function mutation did not. d, Top Venn diagram showing that the difference between the adult lin-4 null neuronal transcriptome compared to the adult control(Ctl) neuronal transcriptome is largely recapitulated in the transcriptome of adult lin-14(ot1149) gf mutants. Only one gene is significantly different in the adult lin-28(ot1154) gf vs adult control comparison and does not overlap with the genes regulated by lin-4/lin-14. Bottom left Venn diagram showing that 48% of genes that demonstrate developmental upregulation (adult control(Ctl)>L1 Ctl) are juvenized in the adult lin-4 null and/or lin-14(ot1149) gf animals. Bottom right Venn diagram showing that 33% of genes that demonstrate developmental downregulation (adult control(Ctl)<L1 Ctl) are juvenized in the adult lin-4 null and/or lin-14(ot1149) gf animals. e, lin-4 regulates lin-28 mainly through lin-14 and not through direct repression of lin-28. On the top left is the schematic of the regulation between lin-4, lin-14 and lin-28 based upon previous studies. On the top right is the schematic of the lin-28 translational GFP allele, as engineered by CRISPR-Cas9, as well as lin-28(ot1153/54/55) gf and lin-28(n719) loss-of-function (lf) alleles. These three gf alleles represent the same molecular lesion (deletion of single lin-4 binding site in the lin-28 3’UTR) but independent CRISPR-Cas9 mediated deletion events. On the bottom are the representative images of the lin-28 translational GFP allele across post-embryonic development in control, lin-4(e912) null, lin-14(ot1151) gf, and lin-28(ot1153) gf animals. The signal is diffuse and cytoplasmic but can be observed in all tissues including the nervous system in early larval animals. Red scale bars (10µm) are on the bottom right of all representative images. LIN-28 is downregulated across post-embryonic development. lin-4(e912) null and lin-14 (ot1151) gf mutations delay the downregulation of LIN-28, particularly during the L2->L3 transition, while the lin-28(ot1153) gf mutation does not. f, lin-28 does not regulate developmental expression pattern of nlp-45. Representative images of the nlp-45 expression reporter in control, lin-28(n719) lf and lin-28(1155) gf animals. Neurons that are labelled in black are not developmentally regulated while those that are labelled in red are developmentally upregulated. Red scale bars (10µm) are on the bottom right of all representative images.

Extended Data Fig. 7 Developmentally regulated genes not controlled by lin-4/lin-14.

a, ins-6 developmental expression is not regulated by lin-4 nor lin-14. Representative images of the ins-6 expression reporter in control, lin-4 null and lin-14 null animals are shown on the left while quantification of the ins-6 expression in ASJ is shown on the right (number of animals for each condition is shown in red brackets). b, ins-9 developmental expression is not regulated by lin-4. Representative images of the ins-9 expression reporter in control and lin-4 null animals are shown on the left while quantification of the ins-9 expression is shown on the right (number of animals for each condition is shown in red brackets). c, inx-2 developmental expression is not regulated by lin-4. Representative images of the inx-2 expression reporter in control and lin-4 null animals are shown on the left while quantification of neuronal inx-2 expression is shown on the right (number of animals for each condition is shown in red brackets). d, flp-26 developmental expression is not regulated by lin-4. Representative images of the flp-26 expression reporter in control and lin-4 null animals are shown. Control images for A-D are taken from Extended Data Fig. 3. Red scale bars (10µm) are on the bottom right of all representative images.

Extended Data Fig. 8 Decreased LIN-14 binding across L1->L2 transition controls lin-4/lin-14 dependent transcriptomic changes.

a, Decreased LIN-14 binding at promoters of target genes during the L1 -> L2 transition. Normalized datasets at each developmental stage against respective inputs are generated using the bamCompare function of deepTools255. LIN-14 enrichment around TSS +/− 2kb is then computed using the computeMatrix function and plotted using the plotHeatmap function of deepTools255. b, LIN-14 ChIP–seq peak distribution and motif across L1 and L2 animals. The ChIP–seq peak distribution on the left is plotted using ChIPseeker48. The consensus binding motif on the right is obtained using MEME-ChIP49. c, Amalgamation of different methods of assessing differential LIN-14 binding across the L1->L2 transition. Outer three Venn Diagrams on the top are the 32990 LIN-14 peaks in L1 animals within 3kb of the TSS of 18245 genes (green, Supplementary Table 8), the 7240 LIN-14 peaks in L2 animals within 3kb of the TSS of 5818 genes (red, Supplementary Table 9), and the 5267 differential L1 vs L2 LIN-14 binding within 3kb of the TSS of 4532 genes (blue, as determined by DiffBind, Supplementary Table 10), each overlapped with the 7974 neuronally enriched genes. These neuronally enriched peaks in L1 (green), peaks in L2 (red) and differential L1 vs L2 binding (blue) were overlapped in the middle Venn Diagram to result in the 3466 genes (Supplementary Table 11) that show differential (mostly decreased) L1 vs L2 binding. These 3466 genes overlapped with 339 (60%) of the lin-4/lin-14 controlled developmentally upregulated genes and 138 (49%) of the lin-4/lin-14 controlled developmentally downregulated genes. d, The 339 genes that showed developmental upregulation (LIN-14 as a repressor) had increased LIN-14 peak enrichment and number of LIN-14 peaks within 3kb of TSS as compared to the 138 genes that showed developmental downregulation (LIN-14 as an activator) or the 2989 genes that show no developmental regulation. Box Whisker plots (10-90 percentile) are used, and two-sided t-test with post hoc correction P values are shown for the comparisons. e, Heat map of the developmental up-/down-regulated genes that also had differential LIN-14 binding from c across development (L1 and adult) and genotype (Control[Ctl] and lin-4/lin-14 mutants). Values were z-score normalized and plotted using pheatmap in R studio. Each row represents a single gene, and each column represents a single RNA-seq replicate. The rows are clustered according to gene expression patterns.

Extended Data Fig. 9 lin-4/lin-14 control developmental regulation of neuropeptide-encoding and receptor-type guanylyl cyclase (rGC) genes.

For all panels, the plots with normalized RNA-seq read counts for the L1/adult control/heterochronic mutant animals are plotted, with each point representing a replicate (n in bracket) and the mean +/- SEM shown for each stage, on the upper left. Adjusted P values (Padj), as calculated by DESeq2, for each comparison are below. lin-4 null/lin-14 gain-of-function mutations juvenize the expression of all four genes. LIN-14 ChIP–seq binding at each gene is shown on the upper right. All samples from their respective experimental conditions are merged for their respective tracks in IGV. Decrease in LIN-14 binding during the L1->L2 transition is observed for all 4 genes. Representative confocal images under control and mutant conditions are shown in the bottom left. d in brackets denote dim expression, while v in brackets denote variable expression. Red scale bars (10µm) are on the bottom right of all representative images. Quantification of the images are shown on the bottom right (number of animals for each condition is shown in red brackets). a, Juvenization of nlp-45 expression by lin-4/lin-14 across development as predicted from the neuronal INTACT/RNA-seq profiling and LIN-14 ChIP–seq binding at the nlp-45 gene. Representative images are shown in Fig. 3a. b, flp-28 expression is gained in hermaphrodite specific neurons (HSN, VC) during transition into late larval/adult stages while the same developmental upregulation is not observed in lin-4 null mutants. flp-28 expression in head/tail neurons is not regulated in lin-4 null animals (images not shown). Control images are taken from Extended Data Fig. 3f. c, flp-14 loses and gains expression in one (LUA, outlined in green) and two (AVB and AVG, outlined in red) classes of neurons as it enters late larval/adult stages, respectively. flp-14 expression is de-repressed in the AVB and AVG neurons in L1 lin-14 null animals while flp-14 expression is repressed in the LUA neurons in L1 lin-14 null animals. Consistently, flp-14 expression in the AVB and AVG neurons are repressed in adult lin-4 null animals while flp-14 expression is increased in the LUA neuron as compared to adult control animals. flp-14 expression is also weakly de-repressed in two classes of neurons that express flp-14 in the adult male (RMDD/V, outlined in blue) and one additional class of neuron (AVE, outlined in purple) in L1 lin-14 null animals. Control images are taken from Extended Data Fig. 3i. d, gcy-12 gains expression in the A and B type motor neurons across mid/late larval development. gcy-12 expression in the A and B type motor neurons is de-repressed in lin-14 null L1 animals while gcy-12 expression in the A and B type motor neurons is repressed in the lin-4 null adult animals as compared to respective control animals. gcy-12 expression in head/tail neurons is not regulated in lin-4 null animals (images not shown). e, nlp-13 gains expression in the ventral nerve cord neurons (DA, VA, VD, VC) across development. nlp-13 expression in the DA neurons is de-repressed in lin-14 null L1 animals as compared to L1 control animals. Control L1 and adult images are taken from Extended Data Fig. 3j.

Extended Data Fig. 10 Regulation and function of nlp-45 across temporal, sexual, and environmental dimensions of post-embryonic development.

a, Schematic of nlp-45 deletion mutants and exploratory assay. b, Increased dwelling during L1->L2 transition is partially juvenized in lin-4(e912) animals. Mean +/- SEM and n (in bracket) are shown for each condition, and each point of the scatter dot plot represents a single animal. Wilcoxon rank-sum tests and false-discovery rate q values for each comparison shown below. c, Schematic showing lin-4/lin-14 regulation of nlp-45 to alter exploratory behaviour during the L1>L2 transition. d, nlp-45 deletion mutants do not significantly affect the food leaving behaviours of juvenile males/hermaphrodites. Values were plotted as mean +/- SEM of three independent experiments (n=6 animals per independent experiments). e, Leaving assay for adult hermaphrodite in nlp-45 and pdf-1 mutant animals. Values were plotted as mean +/- SEM of three independent experiments (n= 8 animals per independent experiments). Statistical analysis (two-sided t-test with post hoc correction) is only shown for the comparison to respective controls (colour coded respectively). f, Leaving assay for adult male in nlp-45 and pdf-1 mutant animals. Values plotted as mean +/- SEM of three independent experiments (n= 8 animals per independent experiments). Statistical analysis (two-sided t-test with post hoc correction) is only shown for the comparison to respective controls (colour coded respectively). g, Schematic of the opposing role of nlp-45 and pdf-1 on male food leaving behaviour. h, Developmental expression of lin-14 in hermaphrodites and males. Representative images for the lin-14(cc2841[lin-14::gfp]) reporter are shown across all developmental stages for both sexes. LIN-14 expression was similarly downregulated in both sexes at early larval stages, its expression in the late larval and particularly in the adult stage was significantly more reduced in the male nervous system compared to that of the hermaphrodite. Ellipse and polygon outline anterior and lateral/ventral neuronal ganglia. Representative images for hermaphrodite are re-used here from Extended Data Fig. 5b for direct side by side comparison with male animals across development. i, Pan-neuronal depletion of sex determination master regulator TRA-1, through overexpression of FEM-3, decreases nervous system LIN-14 expression in adult hermaphrodites to mimic that of adult males. Representative microscope images, shown above, are overexposed in comparison to previous lin-14 reporter images to better show the dim expression in adult males and FEM-3 overexpressed hermaphrodites. The quantifications of head neuron numbers across the three conditions are shown below. The mean +/- SEM and n (in bracket) shown for each condition, and each point of the scatter dot plot represents a single animal. Two-sided t-test with post hoc correction P values are shown for each comparison. j, Pan-neuronal depletion of TRA-1, through overexpression of FEM-3, masculinizes nlp-45 expression in adult hermaphrodite VNC. Representative images are shown on the left. Quantifications of VNC neuron numbers are shown on the right. The mean +/- SEM and n (in bracket) are shown for each condition, and each point of the scatter dot plot represents a single animal. Two-sided t-test with post hoc correction P values are shown for each comparison. k, Sexually dimorphic expression of flp-14 in adult hermaphrodites and males. In addition to stronger flp-14 expression in the AVB neuron (red) as compared to adult hermaphrodites, adult males gain flp-14 expression in the RMDD/V and SIA neurons (blue). Adult hermaphrodite images are re-used from Extended Data Fig. 3i. l, Sexually dimorphic expression of flp-28 in adult hermaphrodites and males. As compared to adult hermaphrodites, adult males gain flp-28 expression in the IL1D/V, URB, and AIM neurons (blue). Adult hermaphrodite images are re-used from Extended Data Fig. 3f. m, Sexually dimorphic expression of nlp-13 in adult hermaphrodites and males. In addition to stronger nlp-13 expression in the VD neurons (red) as compared to adult hermaphrodites, adult males gain nlp-13 expression in the male specific CA/CP motor neurons (blue) and lose nlp-13 expression in the DA, VA and hermaphrodite specific VC motor neurons. Adult hermaphrodite images are re-used from Extended Data Fig. 3j. n, Pan-neuronal degradation of DAF-16 in auxin-treated dauers leads to pan-neuronal de-repression of lin-14 in dauer animals. Representative images are on the left while binary quantifications of pan-neuronal expression are shown on the right (number of animals for each condition is shown in red brackets). Animals were grown (from embryo onward) on NGM plates supplemented with OP50 and 4mM auxin in EtOH (indole-3 acetic acid, IAA, Alfa Aesar) at 25 °C to degrade DAF-16 pan-neuronally and to induce dauer formation. As controls, plates were supplemented with the solvent EtOH instead of auxin. Additional control animals without pan-neuronal TIR-1 expression grown on EtOH and auxin were also included for comparison. o, Pan-neuronal degradation of DAF-16 in auxin-treated dauers leads to a loss or reduced nlp-45 expression in several neuronal classes. Representative images are shown in Fig. 4f. Binary quantifications are shown for the SAAD/V neurons (number of animals for each condition is shown in red brackets) while fluorescence quantifications are shown for the RMED/V, RMEL/R, IL1D/V, RIV, RIM, and ADE neurons. The mean +/- SEM and n (in bracket) are shown for each condition, and each point of the scatter dot plot represents a single animal. Two-sided t-test with post hoc correction P values are shown for each comparison. p, Expression of flp-14 in dauer animals as compared to L3 animals. Upon entry into dauer, similar to expression pattern in the adult hermaphrodite/male, animals gain flp-14 expression in the AVB neurons (red) and lose flp-14 expression in the LUA neurons (green). Additional dauer specific flp-14 expression is gained in the ASE neurons (orange). Expression in the PVR neurons is also lost in dauer animals. L3 hermaphrodite images are re-used from Extended Data Fig. 3i. q, Expression of flp-28 in dauer animals as compared to L3 animals. Upon entry into dauer, animals gain flp-28 expression in the ALA, AVH, AIN, ADA, and DVA neurons (labelled in orange) and lose flp-28 expression in the DVC neurons. L3 hermaphrodite images are re-used from Extended Data Fig. 3f. d and v in brackets denote dim and variable expression, respectively. Scale bars = 10µm.

Extended Data Fig 11 Terminal selector provides spatial specificity to nlp-45 expression pattern.

a, Regulation of nlp-45 by cell specific regulator, unc-42. Representative images of adult unc-42(e419) hermaphrodite and male animals are shown on the left while binary quantifications of nlp-45 expression in the RMDD/V, RIV, SAAD/V and AVA neurons are shown on the right (number of animals for each condition is shown in red brackets). nlp-45 expression was lost in all unc-42 expressing neurons with the exception of AVA in unc-42 mutant animals. b, Regulation of nlp-45 by cell specific regulator, unc-3. Representative images of adult unc-3(e151) hermaphrodite and male animals are shown on the left while binary quantifications of nlp-45 expression in the SAAD/V and AVA neurons are shown in the middle (number of animals for each condition is shown in red brackets). The quantification of ventral nerve cord (VNC) motor neurons is shown on the right. n (in bracket) and two-sided t-test with post hoc correction P values are shown. Each point of the scatter dot plot represents a single animal. nlp-45 expression was lost in unc-3 expressing head neurons (i.e. SAAD/V, AVA) while severely affected in the VNC in unc-3 mutant animals. c, Regulation of nlp-45 by cell specific regulator, ast-1. Representative images of adult ast-1(ot417) hypomorph hermaphrodite and male animals are shown on the left while quantifications of nlp-45 expression in the CEPD and CEPV neurons are shown on the right (number of animals for each condition is shown in red brackets). nlp-45 expression was severely affected in the CEPD neurons and slightly affected in the CEPV neurons in the ast-1 mutant animals. d, Regulation of nlp-45 by cell specific regulator, ceh-8. Representative images of adult ceh-8(gk116531) hermaphrodite and male animals are shown on the left while binary quantifications of nlp-45 expression in the RIA and AVE neurons are shown on the right (number of animals for each condition is shown in red brackets). nlp-45 expression is lost in the RIA neurons and ectopically gained in the AVE neurons in ceh-8 mutant animals. e, Terminal selector (unc-42) does not regulate heterochronic pathway (lin-14). On the left are representative images of L1 lin-14 translational GFP allele worms in control and unc-42(e419) backgrounds. On the right is the quantification of fluorescence intensity in the lateral/ventral ganglion of control vs unc-42(e419) L1 animals. The mean +/- SEM and n (in bracket) are shown for each condition, and each point of the scatter dot plot represents a single animal. f, Heterochronic pathway (lin-14) does not regulate terminal selector (unc-42). Representative images of L1 unc-42 translational GFP allele worms in control and lin-14(ma135) null backgrounds are shown. g, On top are representative images of the nlp-45 expression reporter in control, lin-14(0), and lin-14(0); unc-42(0) L1 animals. On the bottom is are the binary quantifications of nlp-45 expression in different neuronal subtypes in control, lin-14(0), and lin-14(0); unc-42(0) L1 animals (number of animals for each condition is shown in red brackets). nlp-45 showed precocious expression in lin-14, unc-42 double mutants, similar to the lin-14 null mutant alone, except in the neurons (i.e. RMDD/V, SAAD/V) where unc-42 acts as a terminal selector.

Supplementary information

Supplementary Information

Supplementary Discussion and References, Supplementary Fig. 1 and the legends for Supplementary Tables 1–13.

Reporting Summary

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Supplementary Table 1

Worm tracking summary. The mean, s.d. and s.e.m. for each of the 726 parameters at each developmental stage are shown. The statistics (q(Wilcoxon)) for each parameter and each comparison are also shown.

Supplementary Table 2

Neuronally enriched genes. Comparison of neuronal nuclei IP samples to input (total nuclei) samples was conducted using DESeq2 (ref. 42) in R Studio to determine the enrichment; 7,974 genes have a log2[fold change] > 0 (neuronally enriched over input) and an adjusted P (Padj) < 0.05. The genes are sorted by Padj from the smallest to largest. baseMean is the average normalized read counts of all IP and input samples. log2[fold change] was calculated using the formula log2[average read counts of IP samples/average read counts of input samples].

Supplementary Table 3

Normalized read counts of all 7,974 neuronally enriched genes across post-embryonic development. Raw read count for the 7,974 neuronally enriched genes are extracted for the neuronal IP samples and adjusted for library size. These are then used in DESeq2 (ref. 42) to conduct comparisons between developmental stages, as shown in Supplementary Tables 4 and 5. The final read counts displayed are as a result of normalization performed using DESeq2. There are 4, 5, 6 and 7 replicates shown for the L1, L2, L4 and adult stages, respectively.

Supplementary Table 4

Comparison of all temporal transitions among the 7,974 neuronally enriched genes across post-embryonic development. Pairwise comparisons between all stages are conducted using DESeq2 (ref. 42) as described in Supplementary Table 2. The log2[fold change] and Padj are shown for each comparison. All 7,974 neuronally enriched genes are shown regardless of Padj. The table is sorted alphabetically by gene name.

Supplementary Table 5

Normalized read counts of 2,639 developmentally regulated genes. The same as Supplementary Table 3, but with a subset of only 2,639 genes with Padj < 0.01 in any pairwise comparisons between developmental stages.

Supplementary Table 6

Developmental change summary. Expression patterns, at the single-neuron resolution, for all of the validated reporters from Extended Data Figs. 2–4 are displayed here. The neurons that show expression for each gene at each post-embryonic developmental stage (L1 to adult) are listed. Neurons in black do not show altered developmental expression patterns, whereas those labelled in green and red show decreases and increases in gene expression across development, respectively. Those labelled in brown demonstrate both increases and decreases in expression across development. Dim expression (d), moderate level of expression (m), and variable expression (v) are indicated.

Supplementary Table 7

Genes that are juvenized in adult heterochronic mutants. Pairwise comparisons between adult lin-4(e912) null, lin-14(ot1149) gain-of-function (gf) and lin-28(ot1154) gain-of-function mutant neuronal IPs are conducted against adult control neuronal IP data, using only the read counts of the 7,974 neuronally enriched genes. The log2[fold change] and Padj are shown for each comparison. All 7,974 neuronally enriched genes are shown regardless of Padj. The table is sorted alphabetically by gene name. log2[fold change] is calculated using the formula log2[average read counts of mutant adult samples/average read counts of control adult samples].

Supplementary Table 8

LIN14 ChIP peaks in L1 animals. The five replicates from L1 animals are merged and the peaks were called using MACS2 (ref. 46). The ChIP–seq peaks were annotated to the nearest gene using ChIPseeker (ref. 47). Only the peaks within 3 kb of the closest genes were retained for this table. The peaks are arranged alphabetically by the name of the closest associated gene.

Supplementary Table 9

LIN14 ChIP peaks in L2 animals. The five replicates from L2 animals are merged and the peaks were called using MACS2 (ref. 46). The ChIP–seq peaks were annotated to the nearest gene using ChIPseeker47. Only the peaks within 3 kb of the closest genes were retained for this table. The peaks were arranged alphabetically by the name of the closest associated gene.

Supplementary Table 10

Differential LIN14 ChIP peaks between L1 and L2 animals as determined using DiffBind. Differential binding analysis between L1 and L2 samples was performed using Diffbind49. All of the differential binding sites are annotated and assigned to the nearest gene using ChIPseeker47. Only the peaks within 3 kb of the closest genes were retained for this table. The peaks were arranged alphabetically by the name of the closest associated gene.

Supplementary Table 11

List of genes that exhibit differential LIN14 binding during the L1-to-L2 transition. This table contains the 3,466 genes that show differential (mostly decreased) L1 versus L2 binding. This list was obtained using an amalgamation of different methods of assessing differential LIN-14 binding across the L1-to-L2 transition, as described in Extended Data Fig. 8c. The genes are sorted alphabetically.

Supplementary Table 12

Sexually dimorphic and dauer-induced expression patterns of LIN-4/LIN-14 controlled developmentally regulated genes. The sexually dimorphic and dauer-induced expression patterns of five genes (nlp-45, flp-28, flp-14, gcy-12 and nlp-13) that demonstrate LIN-4/LIN-14 controlled developmental regulation are summarized in this table (for representative images, see Fig. 3a and Extended Data Fig. 9). For each gene, the neurons of which the developmental regulation is controlled by LIN-4/LIN-14, are listed in the second column. Those labelled in green and red show decreases and increases in gene expression across post-embryonic development in hermaphrodite animals, respectively. Those labelled in blue gain expression in adult male animals, whereas those in orange show additional expression after entry into dauer. Those labelled in purple are regulated by LIN-4/LIN-14 but are not observed in any conditions tested in control animals. The neurons in which LIN-14 acts as a repressor or an activator are listed in the third and fourth column, respectively. The last three columns show the neurons for each gene of which expression is regulated across post-embryonic development in hermaphrodites, between adult males and hermaphrodites, and between dauer and the comparable L3 mid-larval animals, respectively. For most nlp-45 expressing neurons and several flp-14 expressing neurons, the sexually dimorphic and dauer-specific expression patterns are consistent with regulation through LIN-14. For flp-28, the hash (#) symbol indicates the observation that these neurons, which show increased expression in male and dauer animals, are consistent with the model that these expression patterns are regulated through lin-14. However, due to the proximity of flp-28 and lin-14 locus, the flp-28 expression pattern could not be examined in lin-14 null animals to determine the full battery of neurons that demonstrate lin-14 regulated flp-28 expression. Asterisks indicate that sexually dimorphic or dauer-specific expression patterns of flp-14 for these neurons are not regulated through lin-14. The single plus (+) symbols indicate that no obvious sexually dimorphic and dauer-specific expression patterns were observed for gcy-12. This could be due to the type of reporter (promoter fusion) and the diffuse (cytoplasmic) signal of the reporter. Additional regulatory mechanisms that antagonize lin-14 regulation of gcy-12 could also explain the lack of sexually dimorphic or dauer specific expression pattern of gcy-12 in the ventral nerve cord motor neurons or elsewhere. The double-plus (++) symbols indicate that, for nlp-13, the sexually dimorphic and dauer-specific (no difference) expression patterns are not consistent with lin-14 regulation alone. This suggests that there are additional lin-14-independent regulatory mechanisms in male and dauer animals. Together, these data suggest that for some genes, regulation through lin-14 can largely explain the developmental, sexually dimorphic and dauer-specific expression patterns. However, for other genes regulated by LIN-14, there are probably additional lin-14-independent mechanisms that either synergize with or antagonize the regulation by LIN-14, leading to the complex expression patterns observed across temporal, sexual and environmental dimensions of post-embryonic development.

Supplementary Table 13

Strains Used in this manuscript.

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Sun, H., Hobert, O. Temporal transitions in the post-mitotic nervous system of Caenorhabditis elegans. Nature 600, 93–99 (2021). https://doi.org/10.1038/s41586-021-04071-4

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