ccd-5, a novel cdk-5 binding partner, regulates pioneer axon guidance in the ventral nerve cord of Caenorhabditis elegans.

During nervous system development, axons navigate complex environments to reach synaptic targets. Early extending axons must interact with guidance cues in the surrounding tissue, while later extending axons can interact directly with earlier "pioneering" axons, "following" their path. In Caenorhabditis elegans, the AVG neuron pioneers the right axon tract of the ventral nerve cord. We previously found that aex-3, a rab-3 guanine nucleotide exchange factor, is essential for AVG axon navigation in a nid-1 mutant background and that aex-3 might be involved in trafficking of UNC-5, a receptor for the guidance cue UNC-6/netrin. Here, we describe a new gene in this pathway: ccd-5, a putative cdk-5 binding partner. ccd-5 mutants exhibit increased navigation defects of AVG pioneer as well as interneuron and motor neuron follower axons in a nid-1 mutant background. We show that ccd-5 acts in a pathway with cdk-5, aex-3, and unc-5. Navigation defects of follower interneuron and motoneuron axons correlate with AVG pioneer axon defects. This suggests that ccd-5 mostly affects pioneer axon navigation and that follower axon defects are largely a secondary consequence of pioneer navigation defects. To determine the consequences for nervous system function, we assessed various behavioral and movement parameters. ccd-5 single mutants have no significant movement defects, and nid-1 ccd-5 double mutants are less responsive to mechanosensory stimuli compared with nid-1 single mutants. These surprisingly minor defects indicate either a high tolerance for axon guidance defects within the motor circuit and/or an ability to maintain synaptic connections among commonly misguided axons.

Whole Genome Sequencing identifies reciprocal translocation hT2(I;III) breakpoints.

We used whole-genome sequencing (WGS) data from a Caenorhabditis elegans strain homozygous for the reciprocal translocation hT2(I;III) to identify its breakpoints molecularly. The translocation structure is fairly straightforward, with only minor secondary rearrangement in addition to the primary breakpoints. The graphical representation below depicts the two hT2 half-translocations for ease of conceptualization.

Sequenced Breakpoints of Crossover Suppressor/Inversion qC1.

We used whole-genome sequencing (WGS) data from a number of balanced lethal strains in Caenorhabditis elegans to show that the crossover suppressor qC1 is an inversion. The rearrangement is complex, with a large primary inversion that contains several other smaller inverted regions. The graphical representation below depicts these various qC1 rearrangements for ease of conceptualization. It is the simplest chromosomal structure compatible with the data currently available, but even then it is worth noting that the complexity of the qC1 chromosome can make the graphical reconstruction difficult to understand, and it may seem a bit like relativity theory or artwork from M.C. Escher (https://moa.byu.edu/m-c-eschers-relativity/).

Identification of essential genes in Caenorhabditis elegans through whole-genome sequencing of legacy mutant collections.

It has been estimated that 15%-30% of the ∼20,000 genes in C. elegans are essential, yet many of these genes remain to be identified or characterized. With the goal of identifying unknown essential genes, we performed whole-genome sequencing on complementation pairs from legacy collections of maternal-effect lethal and sterile mutants. This approach uncovered maternal genes required for embryonic development and genes with apparent sperm-specific functions. In total, 58 putative essential genes were identified on chromosomes III-V, of which 52 genes are represented by novel alleles in this collection. Of these 52 genes, 19 (40 alleles) were selected for further functional characterization. The terminal phenotypes of embryos were examined, revealing defects in cell division, morphogenesis, and osmotic integrity of the eggshell. Mating assays with wild-type males revealed previously unknown male-expressed genes required for fertilization and embryonic development. The result of this study is a catalog of mutant alleles in essential genes that will serve as a resource to guide further study toward a more complete understanding of this important model organism. As many genes and developmental pathways in C. elegans are conserved and essential genes are often linked to human disease, uncovering the function of these genes may also provide insight to further our understanding of human biology.

Peel-1 negative selection promotes screening-free CRISPR-Cas9 genome editing in Caenorhabditis elegans.

Improved genome engineering methods that enable automation of large and precise edits are essential for systematic investigations of genome function. We adapted peel-1 negative selection to an optimized Dual-Marker Selection (DMS) cassette protocol for CRISPR-Cas9 genome engineering in Caenorhabditis elegans and observed robust increases in multiple measures of efficiency that were consistent across injectors and four genomic loci. The use of Peel-1-DMS selection killed animals harboring transgenes as extrachromosomal arrays and spared genome-edited integrants, often circumventing the need for visual screening to identify genome-edited animals. To demonstrate the applicability of the approach, we created deletion alleles in the putative proteasomal subunit pbs-1 and the uncharacterized gene K04F10.3 and used machine vision to automatically characterize their phenotypic profiles, revealing homozygous essential and heterozygous behavioral phenotypes. These results provide a robust and scalable approach to rapidly generate and phenotype genome-edited animals without the need for screening or scoring by eye.

PhenoMIP: High-Throughput Phenotyping of Diverse Caenorhabditis elegans Populations via Molecular Inversion Probes.

Whether generated within a lab setting or isolated from the wild, variant alleles continue to be an important resource for decoding gene function in model organisms such as Caenorhabditis elegans With advances in massively parallel sequencing, multiple whole-genome sequenced (WGS) strain collections are now available to the research community. The Million Mutation Project (MMP) for instance, analyzed 2007 N2-derived, mutagenized strains. Individually, each strain averages ∼400 single nucleotide variants amounting to ∼80 protein-coding variants. The effects of these variants, however, remain largely uncharacterized and querying the breadth of these strains for phenotypic changes requires a method amenable to rapid and sensitive high-throughput analysis. Here we present a pooled competitive fitness approach to quantitatively phenotype subpopulations of sequenced collections via molecular inversion probes (PhenoMIP). We phenotyped the relative fitness of 217 mutant strains on multiple food sources and classified these into five categories. We also demonstrate on a subset of these strains, that their fitness defects can be genetically mapped. Overall, our results suggest that approximately 80% of MMP mutant strains may have a decreased fitness relative to the lab reference, N2 The costs of generating this form of analysis through WGS methods would be prohibitive while PhenoMIP analysis in this manner is accomplished at less than one-tenth of projected WGS costs. We propose methods for applying PhenoMIP to a broad range of population selection experiments in a cost-efficient manner that would be useful to the community at large.

John Sulston (1942-2018): a personal perspective.

John Sulston changed the way we do science, not once, but three times - initially with the complete cell lineage of the nematode Caenorhabditis elegans, next with completion of the genome sequences of the worm and human genomes and finally with his strong and active advocacy for open data sharing. His contributions were widely recognized and in 2002 he received the Nobel Prize in Physiology and Medicine.

CRISPR/Cas9 Methodology for the Generation of Knockout Deletions in Caenorhabditis elegans.

The Caenorhabditis elegans Gene Knockout Consortium is tasked with obtaining null mutations in each of the more than 20,000 open reading frames (ORFs) of this organism. To date, approximately 15,000 ORFs have associated putative null alleles. As there has been substantial success in using CRISPR/Cas9 in C. elegans, this appears to be the most promising technique to complete the task. To enhance the efficiency of using CRISPR/Cas9 to generate gene deletions in C. elegans we provide a web-based interface to access our database of guide RNAs (http://genome.sfu.ca/crispr). When coupled with previously developed selection vectors, optimization for homology arm length, and the use of purified Cas9 protein, we demonstrate a robust and effective protocol for generating deletions for this large-scale project. Debate and speculation in the larger scientific community concerning off-target effects due to non-specific Cas9 cutting has prompted us to investigate through whole genome sequencing the occurrence of single nucleotide variants and indels accompanying targeted deletions. We did not detect any off-site variants above the natural spontaneous mutation rate and therefore conclude that this modified protocol does not generate off-target events to any significant degree in C. elegans We did, however, observe a number of non-specific alterations at the target site itself following the Cas9-induced double-strand break and offer a protocol for best practice quality control for such events.

CRISPR-Cas9 human gene replacement and phenomic characterization in Caenorhabditis elegans to understand the functional conservation of human genes and decipher variants of uncertain significance.

Our ability to sequence genomes has vastly surpassed our ability to interpret the genetic variation we discover. This presents a major challenge in the clinical setting, where the recent application of whole-exome and whole-genome sequencing has uncovered thousands of genetic variants of uncertain significance. Here, we present a strategy for targeted human gene replacement and phenomic characterization, based on CRISPR-Cas9 genome engineering in the genetic model organism Caenorhabditis elegans, that will facilitate assessment of the functional conservation of human genes and structure-function analysis of disease-associated variants with unprecedented precision. We validate our strategy by demonstrating that direct single-copy replacement of the C. elegans ortholog (daf-18) with the critical human disease-associated gene phosphatase and tensin homolog (PTEN) is sufficient to rescue multiple phenotypic abnormalities caused by complete deletion of daf-18, including complex chemosensory and mechanosensory impairments. In addition, we used our strategy to generate animals harboring a single copy of the known pathogenic lipid phosphatase inactive PTEN variant (PTEN-G129E), and showed that our automated in vivo phenotypic assays could accurately and efficiently classify this missense variant as loss of function. The integrated nature of the human transgenes allows for analysis of both homozygous and heterozygous variants and greatly facilitates high-throughput precision medicine drug screens. By combining genome engineering with rapid and automated phenotypic characterization, our strategy streamlines the identification of novel conserved gene functions in complex sensory and learning phenotypes that can be used as in vivo functional assays to decipher variants of uncertain significance.

Mutational and transcriptional landscape of spontaneous gene duplications and deletions in Caenorhabditis elegans.

Gene duplication and deletion are pivotal processes shaping the structural and functional repertoire of genomes, with implications for disease, adaptation, and evolution. We employed a mutation accumulation (MA) framework partnered with high-throughput genomics to assess the molecular and transcriptional characteristics of newly arisen gene copy-number variants (CNVs) in Caenorhabditis elegans populations subjected to varying intensity of selection. Here, we report a direct spontaneous genome-wide rate of gene duplication of 2.9 × 10-5/gene per generation in C. elegans, the highest for any species to date. The rate of gene deletion is sixfold lower (5 × 10-6/gene per generation). Deletions of highly expressed genes are particularly deleterious, given their paucity in even the N = 1 lines with minimal efficacy of selection. The increase in average transcript abundance of new duplicates arising under minimal selection is significantly greater than twofold compared with single copies of the same gene, suggesting that genes in segmental duplications are frequently overactive at inception. The average increase in transcriptional activity of gene duplicates is greater in the N = 1 MA lines than in MA lines with larger population bottlenecks. There is an inverse relationship between the ancestral transcription levels of new gene duplicates and population size, with duplicate copies of highly expressed genes less likely to accumulate in larger populations. Our results demonstrate a fitness cost of increased transcription following duplication, which results in purifying selection against new gene duplicates. However, on average, duplications also provide a significant increase in gene expression that can facilitate adaptation to novel environmental challenges.

The Molecular Chaperone HSP90 Promotes Notch Signaling in the Germline of Caenorhabditis elegans.

In a genetic screen to identify genes that promote GLP-1/Notch signaling in Caenorhabditis elegans germline stem cells, we found a single mutation, om40, defining a gene called ego-3. ego-3(om40) causes several defects in the soma and the germline, including paralysis during larval development, sterility, delayed proliferation of germline stem cells, and ectopic germline stem cell proliferation. Whole genome sequencing identified om40 as an allele of hsp-90, previously known as daf-21, which encodes the C. elegans ortholog of the cytosolic form of HSP90. This protein is a molecular chaperone with a central position in the protein homeostasis network, which is responsible for proper folding, structural maintenance, and degradation of proteins. In addition to its essential role in cellular function, HSP90 plays an important role in stem cell maintenance and renewal. Complementation analysis using a deletion allele of hsp-90 confirmed that ego-3 is the same gene. hsp-90(om40) is an I→N conservative missense mutation of a highly conserved residue in the middle domain of HSP-90 RNA interference-mediated knockdown of hsp-90 expression partially phenocopied hsp-90(om40), confirming the loss-of-function nature of hsp-90(om40) Furthermore, reduced HSP-90 activity enhanced the effect of reduced function of both the GLP-1 receptor and the downstream LAG-1 transcription factor. Taken together, our results provide the first experimental evidence of an essential role for HSP90 in Notch signaling in development.

MIP-MAP: High-Throughput Mapping of Caenorhabditis elegans Temperature-Sensitive Mutants via Molecular Inversion Probes.

Mutants remain a powerful means for dissecting gene function in model organisms such as Caenorhabditis elegans Massively parallel sequencing has simplified the detection of variants after mutagenesis but determining precisely which change is responsible for phenotypic perturbation remains a key step. Genetic mapping paradigms in C. elegans rely on bulk segregant populations produced by crosses with the problematic Hawaiian wild isolate and an excess of redundant information from whole-genome sequencing (WGS). To increase the repertoire of available mutants and to simplify identification of the causal change, we performed WGS on 173 temperature-sensitive (TS) lethal mutants and devised a novel mapping method. The mapping method uses molecular inversion probes (MIP-MAP) in a targeted sequencing approach to genetic mapping, and replaces the Hawaiian strain with a Million Mutation Project strain with high genomic and phenotypic similarity to the laboratory wild-type strain N2 We validated MIP-MAP on a subset of the TS mutants using a competitive selection approach to produce TS candidate mapping intervals with a mean size < 3 Mb. MIP-MAP successfully uses a non-Hawaiian mapping strain and multiplexed libraries are sequenced at a fraction of the cost of WGS mapping approaches. Our mapping results suggest that the collection of TS mutants contains a diverse library of TS alleles for genes essential to development and reproduction. MIP-MAP is a robust method to genetically map mutations in both viable and essential genes and should be adaptable to other organisms. It may also simplify tracking of individual genotypes within population mixtures.

Mitochondrial Mutation Rate, Spectrum and Heteroplasmy in Caenorhabditis elegans Spontaneous Mutation Accumulation Lines of Differing Population Size.

Mitochondrial genomes of metazoans, given their elevated rates of evolution, have served as pivotal markers for phylogeographic studies and recent phylogenetic events. In order to determine the dynamics of spontaneous mitochondrial mutations in small populations in the absence and presence of selection, we evolved mutation accumulation (MA) lines of Caenorhabditis elegans in parallel over 409 consecutive generations at three varying population sizes of N = 1, 10, and 100 hermaphrodites. The N =1 populations should have a minimal influence of natural selection to provide the spontaneous mutation rate and the expected rate of neutral evolution, whereas larger population sizes should experience increasing intensity of selection. New mutations were identified by Illumina paired-end sequencing of 86 mtDNA genomes across 35 experimental lines and compared with published genomes of natural isolates. The spontaneous mitochondrial mutation rate was estimated at 1.05 × 10-7/site/generation. A strong G/C→A/T mutational bias was observed in both the MA lines and the natural isolates. This suggests that the low G + C content at synonymous sites is the product of mutation bias rather than selection as previously proposed. The mitochondrial effective population size per worm generation was estimated to be 62. Although it was previously concluded that heteroplasmy was rare in C. elegans, the vast majority of mutations in this study were heteroplasmic despite an experimental regime exceeding 400 generations. The frequencies of frameshift and nonsynonymous mutations were negatively correlated with population size, which suggests their deleterious effects on fitness and a potent role for selection in their eradication.

Accelerating Gene Discovery by Phenotyping Whole-Genome Sequenced Multi-mutation Strains and Using the Sequence Kernel Association Test (SKAT).

Forward genetic screens represent powerful, unbiased approaches to uncover novel components in any biological process. Such screens suffer from a major bottleneck, however, namely the cloning of corresponding genes causing the phenotypic variation. Reverse genetic screens have been employed as a way to circumvent this issue, but can often be limited in scope. Here we demonstrate an innovative approach to gene discovery. Using C. elegans as a model system, we used a whole-genome sequenced multi-mutation library, from the Million Mutation Project, together with the Sequence Kernel Association Test (SKAT), to rapidly screen for and identify genes associated with a phenotype of interest, namely defects in dye-filling of ciliated sensory neurons. Such anomalies in dye-filling are often associated with the disruption of cilia, organelles which in humans are implicated in sensory physiology (including vision, smell and hearing), development and disease. Beyond identifying several well characterised dye-filling genes, our approach uncovered three genes not previously linked to ciliated sensory neuron development or function. From these putative novel dye-filling genes, we confirmed the involvement of BGNT-1.1 in ciliated sensory neuron function and morphogenesis. BGNT-1.1 functions at the trans-Golgi network of sheath cells (glia) to influence dye-filling and cilium length, in a cell non-autonomous manner. Notably, BGNT-1.1 is the orthologue of human B3GNT1/B4GAT1, a glycosyltransferase associated with Walker-Warburg syndrome (WWS). WWS is a multigenic disorder characterised by muscular dystrophy as well as brain and eye anomalies. Together, our work unveils an effective and innovative approach to gene discovery, and provides the first evidence that B3GNT1-associated Walker-Warburg syndrome may be considered a ciliopathy.

UBR-5, a Conserved HECT-Type E3 Ubiquitin Ligase, Negatively Regulates Notch-Type Signaling in Caenorhabditis elegans.

Notch-type signaling mediates cell-cell interactions important for animal development. In humans, reduced or inappropriate Notch signaling activity is associated with various developmental defects and disease states, including cancers. Caenorhabditis elegans expresses two Notch-type receptors, GLP-1 and LIN-12. GLP-1 mediates several cell-signaling events in the embryo and promotes germline proliferation in the developing and adult gonad. LIN-12 acts redundantly with GLP-1 in certain inductive events in the embryo and mediates several cell-cell interactions during larval development. Recovery of genetic suppressors and enhancers of glp-1 or lin-12 loss- or gain-of-function mutations has identified numerous regulators of GLP-1 and LIN-12 signaling activity. Here, we report the molecular identification of sog-1, a gene identified in screens for recessive suppressors of conditional glp-1 loss-of-function mutations. The sog-1 gene encodes UBR-5, the sole C. elegans member of the UBR5/Hyd family of HECT-type E3 ubiquitin ligases. Molecular and genetic analyses indicate that the loss of ubr-5 function suppresses defects caused by reduced signaling via GLP-1 or LIN-12. In contrast, ubr-5 mutations do not suppress embryonic or larval lethality associated with mutations in a downstream transcription factor, LAG-1. In the gonad, ubr-5 acts in the receiving cells (germ cells) to limit GLP-1 signaling activity. SEL-10 is the F-box component of SCF(SEL-10) E3 ubiquitin-ligase complex that promotes turnover of Notch intracellular domain. UBR-5 acts redundantly with SEL-10 to limit Notch signaling in certain tissues. We hypothesize that UBR-5 activity limits Notch-type signaling by promoting turnover of receptor or limiting its interaction with pathway components.

Chromoanasynthetic Genomic Rearrangement Identified in a N-Ethyl-N-Nitrosourea (ENU) Mutagenesis Screen in Caenorhabditis elegans.

Chromoanasynthesis is a recently discovered phenomenon in humans with congenital diseases that is characterized by complex genomic rearrangements (CGRs) resulting from aberrant repair of catastrophic chromosomal damage. How these CGRs are induced is not known. Here, we describe the structure and function of dpDp667, a causative CGR that emerged from a Caenorhabditis elegans dauer suppressor screen in which animals were treated with the point mutagen N-ethyl-N-nitrosourea (ENU). dpDp667 comprises nearly 3 Mb of sequence on the right arm of the X chromosome, contains three duplications and one triplication, and is devoid of deletions. Sequences from three out of the four breakpoint junctions in dpDp667 reveal microhomologies that are hallmarks of chromoanasynthetic CGRs. Our findings suggest that environmental insults and physiological processes that cause point mutations may give rise to chromoanasynthetic rearrangements associated with congenital disease. The relatively subtle phenotype of animals harboring dpDp667 suggests that the prevalence of CGRs in the genomes of mutant and/or phenotypically unremarkable animals may be grossly underestimated.

Rapid Increase in frequency of gene copy-number variants during experimental evolution in Caenorhabditis elegans.

BACKGROUND: Gene copy-number variation (CNVs), which provides the raw material for the evolution of novel genes, is widespread in natural populations. We investigated whether CNVs constitute a common mechanism of genetic change during adaptation in experimental Caenorhabditis elegans populations. Outcrossing C. elegans populations with low fitness were evolved for >200 generations. The frequencies of CNVs in these populations were analyzed by oligonucleotide array comparative genome hybridization, quantitative PCR, PCR, DNA sequencing across breakpoints, and single-worm PCR. RESULTS: Multiple duplications and deletions rose to intermediate or high frequencies in independent populations. Several lines of evidence suggest that these changes were adaptive: (i) copy-number changes reached high frequency or were fixed in a short time, (ii) many independent populations harbored CNVs spanning the same genes, and (iii) larger average size of CNVs in adapting populations relative to spontaneous CNVs. The latter is expected if larger CNVs are more likely to encompass genes under selection for a change in gene dosage. Several convergent CNVs originated in populations descended from different low fitness ancestors as well as high fitness controls. CONCLUSIONS: We show that gene copy-number changes are a common class of adaptive genetic change. Due to the high rates of origin of spontaneous duplications and deletions, copy-number changes containing the same genes arose readily in independent populations. Duplications that reached high frequencies in these adapting populations were significantly larger in span. Many convergent CNVs may be general adaptations to laboratory conditions. These results demonstrate the great potential borne by CNVs for evolutionary adaptation.

The C. elegans UNC-23 protein, a member of the BCL-2-associated athanogene (BAG) family of chaperone regulators, interacts with HSP-1 to regulate cell attachment and maintain hypodermal integrity.

Mutations in the unc-23 gene in the free-living nematode, Caenorhabditis elegans result in detachment and dystrophy of the anterior body wall musculature and a bent-head phenotype when grown on solid substrate. We have determined that the unc-23 gene product is the nematode ortholog of the human BAG-2 protein, a member of the Bcl-2 associated athanogene (BAG) family of molecular chaperone regulators. We show that a functional GFP-tagged UNC-23 protein is expressed throughout development in several tissues of the animal, including body wall muscle and hypodermis, and associates with adhesion complexes and attachment structures within these 2 tissues. In humans, the BAG protein family consists of 6 members that all contain a conserved 45 amino acid BAG domain near their C-termini. These proteins bind to and modulate the activity of the ATPase domain of the heat shock cognate protein 70, Hsc70. We have isolated missense mutations in the ATPase domain of the C. elegans heat shock 70 protein, HSP-1 that suppress the phenotype exhibited by unc-23(e25) mutant hermaphrodites and we show that UNC-23 and HSP-1 interact in a yeast-2-hybrid system. The interaction of UNC-23 with HSP-1 defines a role for HSP-1 function in the maintenance of muscle attachment during development.

Remarkably Divergent Regions Punctuate the Genome Assembly of the Caenorhabditis elegans Hawaiian Strain CB4856.

The Hawaiian strain (CB4856) of Caenorhabditis elegans is one of the most divergent from the canonical laboratory strain N2 and has been widely used in developmental, population, and evolutionary studies. To enhance the utility of the strain, we have generated a draft sequence of the CB4856 genome, exploiting a variety of resources and strategies. When compared against the N2 reference, the CB4856 genome has 327,050 single nucleotide variants (SNVs) and 79,529 insertion-deletion events that result in a total of 3.3 Mb of N2 sequence missing from CB4856 and 1.4 Mb of sequence present in CB4856 but not present in N2. As previously reported, the density of SNVs varies along the chromosomes, with the arms of chromosomes showing greater average variation than the centers. In addition, we find 61 regions totaling 2.8 Mb, distributed across all six chromosomes, which have a greatly elevated SNV density, ranging from 2 to 16% SNVs. A survey of other wild isolates show that the two alternative haplotypes for each region are widely distributed, suggesting they have been maintained by balancing selection over long evolutionary times. These divergent regions contain an abundance of genes from large rapidly evolving families encoding F-box, MATH, BATH, seven-transmembrane G-coupled receptors, and nuclear hormone receptors, suggesting that they provide selective advantages in natural environments. The draft sequence makes available a comprehensive catalog of sequence differences between the CB4856 and N2 strains that will facilitate the molecular dissection of their phenotypic differences. Our work also emphasizes the importance of going beyond simple alignment of reads to a reference genome when assessing differences between genomes.

Sarcomeres Pattern Proprioceptive Sensory Dendritic Endings through UNC-52/Perlecan in C. elegans.

Sensory dendrites innervate peripheral tissues through cell-cell interactions that are poorly understood. The proprioceptive neuron PVD in C. elegans extends regular terminal dendritic branches between muscle and hypodermis. We found that the PVD branch pattern was instructed by adhesion molecule SAX-7/L1CAM, which formed regularly spaced stripes on the hypodermal cell. The regularity of the SAX-7 pattern originated from the repeated and regularly spaced dense body of the sarcomeres in the muscle. The extracellular proteoglycan UNC-52/Perlecan linked the dense body to the hemidesmosome on the hypodermal cells, which in turn instructed the SAX-7 stripes and PVD dendrites. Both UNC-52 and hemidesmosome components exhibited highly regular stripes that interdigitated with the SAX-7 stripe and PVD dendrites, reflecting the striking precision of subcellular patterning between muscle, hypodermis, and dendrites. Hence, the muscular contractile apparatus provides the instructive cues to pattern proprioceptive dendrites.