Paaby, Annalise et al. (2011) International Worm Meeting "Uncovering cryptic genetic variation for C. elegans embryogenesis."

Gunsalus, Kris, Rockman, Matt, Piano, Fabio, White, Amelia, Paaby, Annalise

[International Worm Meeting, 2011]

Cryptic genetic variation (CGV) is allelic variation that affects phenotype, but only under certain conditions: when the system is "perturbed" by changes in the environment or genomic background. Such conditional effects are probably common in biological systems, but they pose barriers to the identification of causal alleles that underlie complex traits. In an to effort understand the nature of CGV, we are exploring the genetic architecture of early embryogenesis in C. elegans. Genome-wide screens have identified genes that affect embryogenesis in a single wild-type background (N2), providing a high degree of resolution in our understanding of the genetics underlying this process. We are utilizing this information to knock down embryonic genes in wild isolates, in order to identify natural allelic variants that affect early embryogenesis in perturbed animals. Embryogenesis is normally invariant, but using RNAi to silence critical embryonic genes reveals differences in embryonic lethality across strains. We have used high-throughput phenotyping methods to evaluate differences in hatching across 64 wild C. elegans strains, silenced at 43 different genes. The patterns of lethality indicate significant levels of CGV for embryogenesis. Some genes reveal high variance in lethality, suggesting that these loci are particularly good perturbation targets for revealing CGV elsewhere across the genome. We also observe significant variation in sensitivity to germline RNAi in these worms.

Paaby, Annalise B. et al. (2013) International Worm Meeting "C. elegans harbors pervasive cryptic genetic variation for embryogenesis."

Piano, Fabio, White, Amelia, Paaby, Annalise B., Gunsalus, Kris, Rockman, Matt

[International Worm Meeting, 2013]

To thoroughly explore the genetic architecture of early embryogenesis in C. elegans, we are searching for conditional relationships between embryonic genes. Genome-wide screens have identified genes that affect embryogenesis in a single wild-type background, providing a lot of information about the genetics underlying the process; we are leveraging this information to probe natural genetic variation across many wild C. elegans isolates. We have silenced a suite of 43 critical embryonic genes in 50 wild strains and characterized differences in lethality across strains. We observe pervasive "cryptic genetic variation" (CGV) for embryogenesis---that is, variation within the networks controlling early cell divisions that has functional consequences only under particular conditions. We find that disrupting genes responsible for polarizing the embryo in the first two cell divisions (PAR family members) uncover particularly high levels of CGV. Using association and linkage mapping, we find that very rarely are cryptic variants uncovered by different silenced genes, even among genes that interact closely and produce the same mutant phenotype. These results imply that CGV for embryogenesis has low pleiotropy, and likely point to new components in the embryogenesis gene network.

Wang, Ling et al. (2021) International Worm Meeting "Compensatory evolution in mitochondrial tRNAs"

Paaby, Annalise, Wang, Ling

[International Worm Meeting, 2021]

Deleterious mutations can accumulate in natural populations, and reversal mutations are likely to be rare. However, new mutations at other sites can potentially compensate for prior deleterious genetic changes, reversing fitness. In the same vein, mutations that are neutral at the time of substitution may effectively "pre-compensate" future mutations that would otherwise be deleterious, allowing mutations to accumulate that are deleterious under some conditions. Identifying such compensatory or conditional mutations is difficult, however, without an a priori expectation of sequence function. Here we use the canonical structure-function relationship of the cloverleaf-like tRNA secondary structure, as well as the high mutation rate of tRNAs, to study compensatory evolution. Specifically, we are exploring mutations in mitochondrial tRNAs (mt-tRNAs) in Caenorhabditis nematodes, including interactions between mt-tRNAs and associated factors encoded in the nuclear genome.

Testa, Nicholas et al. (2017) International Worm Meeting "The shape of things to come: elucidating the mechanisms of Developmental System Drift associated with axis formation in Caenorhabditid nematode embryos."

Paaby, Annalise, Testa, Nicholas

[International Worm Meeting, 2017]

Developmental system drift (DSD) occurs when selection for a stable phenotype allows for developmental mechanisms-including genes, pathways, and cellular and sub-cellular processes-to diverge over evolutionary time even as the gross developmental phenotype remains stable across taxa. This phenomenon, first described formally in 2001, has been referenced routinely in the literature and has received some modest theoretical treatment, but little experimental investigation. Here, we investigate DSD and the underlying mechanisms associated with anterior-posterior axis determination and early embryogenesis in 10 nematode species within the genus Caenorhabditis. Caenorhabditid nematodes are an exemplary model of DSD with highly stereotyped and near-invariant early embryonic cell divisions. Previous studies have demonstrated that, despite their near-indistinguishable developmental regime, Caenorhabditid worms may differ in several cellular and developmental phenotypes during early embryogenesis. Furthermore, many of the gene sequences associated with axis determination in early development (especially par-2) have diverged significantly across taxa. In this study, we precisely quantify subtle differences in early embryonic shape and cell-size caused by species-specific differences in axis determination. For each treatment, approximately 30 individual videos of embryogenesis were collected. Young adult worms were dissected, and resulting embryos mounted on agar pads and imaged on a spinning disk confocal DIC microscope; subsequent videos start in the single-cell stage and progress past 4-cells. We then collected images from these videos at precise time points corresponding to both 2-cell and 4-cell embryos. Using geometric morphometrics with a combination of 12 and 18 landmarks/semi-landmarks, we have demonstrated differences in embryonic shape for 2- and 4-cell embryos (respectively) of N2 (elegans) and AF16 (briggsae) at both 20 deg C and 30 deg C. If the underlying mechanisms that regulate embryonic shape remained the same across species, we would expect to see both species react similarly to stress during development. Instead, our data demonstrate that developmental shape trajectories diverge in stressful environments, revealing cryptic genetic variation of their mechanisms. Our data confirm that DSD has occurred between at least two taxa within Caenorhabditis. Future data will include as many as 10 different Caenorhabditid species.

Kaul, Samiksha et al. (2021) International Worm Meeting "Utilizing Quantitative Molecular Techniques to Capture Expression Level Differences in C. elegans Wild Isolates"

Chou , Han Ting, Paaby , Annalise, Kaul, Samiksha

[International Worm Meeting, 2021]

In the C. elegans embryo, a network of interacting proteins are responsible for establishing polarity along the anterior-posterior axis and coordinating the first few cell divisions. This process is very well studied and is used as a model for cell polarity in metazoans. Morphologically, polarity establishment is highly stereotyped and near-invariant across Caenorhabditis and within C. elegans. However, the underlying molecular and genetic pathways show variability, even at the intraspecific level. In this study, we have developed a method to capture expression level differences of proteins involved in polarity establishment across wild isolates of C. elegans. Using microfluidics and single molecule fluorescent in situ hybridization (smFISH), we are able to achieve excellent spatial and temporal resolution for quantification of expression levels in different wild isolates. This ultimately allows us to compare subtle differences in the molecular pathways involved in polarity establishment and maintenance across different strains of C. elegans.

Bell, Avery Davis et al. (2021) International Worm Meeting "Regulatory differences in wild C. elegans strains from investigation of allele-specific expression"

Paaby, Annalise, Bell, Avery Davis, Chou, Han Ting

[International Worm Meeting, 2021]

Across individuals and species, genetic variation leads to variation in gene expression, resulting in further phenotypic variation. One way to understand how genotype regulates gene expression variation is to look within hybrid or heterozygous individuals: any differences in expression from the two alleles within an individual must be caused by cis acting variants located on the same chromosome as the gene of interest, because the global (or trans) environment is shared across the alleles. We have applied such allele-specific expression (ASE) analyses to uncover cis regulated gene expression changes between wild strains of C. elegans and the reference strain N2. Specifically, we performed RNA sequencing on three biological replicates from each of the strains N2, CB4856, EG4348, JU1088, and QX1211, and from F1 crosses between N2 and the other four strains. We performed RNA alignment and quantification using multiple methods to identify optimal ASE analytical frameworks for this species. For example, we mapped RNA reads to the genome and transcriptome; aligned as well as pseudo-aligned reads; and used both the reference genome alone and pseudo-diploid genomes that incorporated the variants from other strains. We further tried multiple methods for gene-level quantification and ASE analysis. Tools and methods investigated included STAR, WASP, Salmon, bowtie2, and EMASE. A preferred analysis identified 2535 genes with ASE - and thus cis regulatory differences - across the strains, with strains with higher nucleotide divergence from N2 having more genes affected by cis regulatory changes, as expected. For example, less-diverged JU1088 showed evidence for differential cis regulation at 255 genes, while more-diverged CB4856 showed evidence at 702 genes. The vast majority of genes with differential cis regulation from N2 showed this differential regulation in only one strain (n = 2263), while 52 genes showed differential regulation vs. N2 in three or all strains. Comparison of ASE differences to expression differences between the parental strains enables characterization of regulatory divergence as in cis vs. in trans across C. elegans. The evolutionary forces underpinning the observed regulatory divergence may be elucidated via associations of regulatory patterns with genome and population genetic parameters. Such characterizations and analyses will be useful in comparing C. elegans regulatory evolution with that of other species and of regulatory evolution across Caenorhabditis species.

Valencia, Francisco et al. (2021) International Worm Meeting "Why do some wild-type strains fail at germline RNAi?"

Alexander, J.C., Valencia, Francisco, Chou, Han Ting, Paaby, Annalise, Deb, Diptodip

[International Worm Meeting, 2021]

The discovery that experimental delivery of dsRNA can induce gene silencing at target genes revolutionized genetics research, both in uncovering essential biological processes and in creating new tools for developmental geneticists. Our ability to silence C. elegans genes by RNA interference (RNAi) arises from the fact that worms induce a native cascade response upon exposure to exogenous dsRNA, a response that we now know is associated with antiviral immunity but also coincides with a suite of processes affecting things like gene regulation and genome defense against transposons. However, previous work has shown that wild-type strains of C. elegans vary dramatically in their response to exogenous RNAi. Here, we investigate why some strains fail to mount a robust RNAi response to germline targets. We observe diversity in mechanism: in some strains, the response is stochastic, absent in most individuals but rapidly turned on in a few; in other strains, the response is consistent but much delayed. Increased activity of the argonaute PPW-1, which is required for germline RNAi in N2, partially rescues the response in some strains, but dampens it further in others. Across strains, we also observe variability in gene expression of known RNAi factors, and strain-specific instances of pseudogenization, gene loss, and allelic divergence. Our results support the conclusions that secondary argonautes share overlapping functions, that a "just right" level of overall activity promotes a robust response, and that the weak germline RNAi we observe in some strains is explained by diverse genetic variants at shared RNAi genes.

Kaul, Samiksha et al. (2022) MicroPubl Biol

Chou, Han Ting, Charles, Seleipiri, Aubry, Guillaume, Lu, Hang, Kaul, Samiksha, Paaby, Annalise B.

[MicroPubl Biol, 2022]

Wild C. elegans strains harbor natural variation in developmental pathways, but investigating these differences requires precise and well-powered phenotyping methods. Here we employ a microfluidics platform for single-molecule FISH to simultaneously visualize the transcripts of three genes in embryos of two distinct strains. We capture transcripts at high resolution by developmental stage in over one hundred embryos of each strain and observe wide-scale conservation of expression, but subtle differences in par-2 and chin-1 abundance and rate of change. As both genes reside in a genomic interval of hyper-divergence, these results may reflect consequences of pathway evolution over long timescales.

White, Amelia G et al. (2011) International Worm Meeting "Rapid and accurate phenotyping of embryonic lethality via image analysis of C. elegans developmental stages from high-throughput image data."

Sontag, Eduardo, Munarriz, Eliana, Cipriani, Patricia G, Kao, Huey-Ling, Piano, Fabio, Gunsalus, Kristin C, Paaby, Annalise, Geiger, Davi, White, Amelia G

[International Worm Meeting, 2011]

We present an automated image analysis system (DevStaR) for quantitative phenotyping of C. elegans embryonic lethality and sterility phenotypes. Our image analysis system counts each developmental stage in an image of a C. elegans population, allowing efficient high throughput calculation of C. elegans viability phenotypes. DevStaR is an object recognition machine comprising several hierarchical layers that build successively more sophisticated representations of the objects (developmental stages) to be classified. The algorithm segments the objects, decomposes the objects into parts, extracts features from these parts, and classifies them using an SVM (support Vector Machine) and global shape information. This enables correct classifications in the presence of complicated occlusions and deformations of the animals. Features of the classified objects are then used to obtain a count of each developmental stage. We are currently using this system to analyze phenotypic data from C. elegans high-throughput genetic screens, and have processed over one million images for lab users so far. Validation of DevStaR measurements will be shown by comparing DevStaR output to both manual counting of developmental stages and manual scores of quantitative phenotypes. DevStaR can provide an accurate measurement of quantitative phenotype and is comparable to manual scoring. DevStaR has been used to score a C. elegans genome wide RNAi screen with up to 30 repeats per clone tested at up to 5 temperatures per clone. The screen consists of over 600,000 images each scored by DevStaR, Analysis of these data illustrate the convenience of DevStaR scoring and the use of a quantitative phenotype. Our system overcomes a previous bottleneck in image analysis by achieving near real-time scoring of image data in a fully automated manner. Our system reduces the need for human evaluation of images and provides rapid quantitative output that is not feasible at high throughput by manual scoring.

Chou, H. et al. (2017) International Worm Meeting "High-Throughput smFISH Analysis to Measure Natural Genetic Variation at Early Developmental Stages."

Deb, D., Paaby, A, Chou, H.

[International Worm Meeting, 2017]

The development of C. elegans is precise and stereotyped, including patterns of cell division in early embryogenesis. Nevertheless, natural genetic variation in wild-type isolates can cause dramatic differences in phenotype following single-gene perturbations, indicating that different wild-type genotypes harbor functional variation in critical gene networks1. These same wild isolates also show extreme variation in the efficacy of germline RNAi1. What are the genetic, molecular and cellular mechanisms that govern these differences? And how do they evolve when stabilizing selection ensures that phenotypic development remains stable and stereotyped? Here we use single-molecule FISH to quantitatively measure the gene expression at specific locations and time points in early development. By characterizing the temporal and spatial heterogeneities of mRNA transcript numbers in the first few cell divisions, we can connect sub-cellular phenotypes to known variations in early embryonic pathway function and germline RNAi. We use a high-throughput, semi-automated pipeline to acquire precise transcript counts at precisely staged embryos, including implementation of the machine learning spot-counting software Aro2. Despite near-invariant cell division phenotypes, wild isolates show significant differences in transcript abundance for critical embryonic genes. These differences in gene expression do not fully explain differences in embryonic lethality following gene knockdown, as neither wild-type gene expression nor transcript abundance following RNAi correlates perfectly with patterns of embryonic lethality. Notably, we observe significant difference in transcript abundance variance following RNAi among wild-type isolates, suggesting inefficiency of RNAi may be controlled by stochastic thresholds. Currently, we are scaling up the experiments using a microfluidic chip specifically designed for worm embryos in order to test hypotheses with high statistical rigor. References: 1- Wild worm embryogenesis harbors ubiquitous polygenic modifier variation. A.B. Paaby, A.G. White, D.D. Riccardi, K.C. Gunsalus, F. Piano, M.V. Rockman. Elife (2015), p. 4 2- Aro: a machine learning approach to identifying single molecules and estimating classification error in fluorescence microscopy images. A.C. Wu and S.A. Rifkin. BMC Bioinformatics (2015), 16:102