ZACHARIAS, AMANDA L. et al. (2015) International Worm Meeting "The rich get richer: comprehensive quantitative analysis of nuclear SYS-1/beta-catenin and POP-1/TCF in C. elegans embryos identifies a novel memory mechanism for gene expression diversification."

Walton, Travis, Preston, Elicia, MURRAY, JOHN ISAAC, Zacharias, Amanda L.

[International Worm Meeting, 2015]

Quantitative differences in signaling pathway activity are a powerful potential mechanism for diversifying cell fates during development. However, few examples of this mechanism have been identified in vivo, in part due to the challenges of performing quantitative assays. The purpose of this study was to evaluate whether C. elegans embryos use quantitative differences Wnt pathway activity to regulate gene expression. The conserved Wnt pathway acts repeatedly during development, transcriptionally regulating distinct targets in different stages and cell types (i.e. contexts). This dependence of targets on context could reflect not only interactions with differentially expressed transcription factors, but also quantitative, context-specific differences in the nuclear localization of the Wnt pathway effector transcription factor, POP-1/TCF and its cofactor SYS-1/beta-catenin. We investigated the role of Wnt pathway activity in target expression by using time-lapse microscopy and automated lineage tracing of Caenorhabditis elegans embryos to quantify expression of Wnt ligands, target genes, and nuclear localization of POP-1 and SYS-1 in vivo at single cell resolution for all cells throughout embryonic development. Contrary to the exiting binary model, we found reproducible quantitative variability in POP-1 and SYS-1 across cells. Nuclear levels of SYS-1 are constant over time but concentrations increase due to decreases in nuclear volume. Cells in which Wnt signaling was activated for two or more consecutive cell cycles have higher nuclear SYS-1 concentration and stronger POP-1-mediated transcriptional activation than cells where the parent was not signaled, indicating a "memory" of previous states. We measured the POP-1-dependence of candidate targets and identified over a dozen important developmental regulators as new POP-1 targets. We found that most targets require POP-1 for either activation or repression, but not both. The targets that require POP-1 for activation are preferentially expressed in the cells where Wnt signaling was active for consecutive cell cycles, consistent with a functional role for the quantitative difference we observed. Taken together, these results suggest that the enrichment of nuclear POP-1 and SYS-1 across mitosis provides a cellular memory mechanism to integrate lineage history and allow POP-1 to activate distinct targets in different developmental contexts. Future identification of POP-1, SYS-1 and context factor binding sites will help further refine our understanding of the mechanisms governing context-specific activity.

Richards, Julia L et al. (2011) International Worm Meeting "Characterizing development by lineage tracing."

Preston, Elicia, Richards, Julia L, Walton, Travis, Burdick, Joshua, Murray, John

[International Worm Meeting, 2011]

We previously developed automated lineage tracing methods and used these methods to characterize the expression pattern of over 120 gene reporters in C. elegans embryos through the 350-cell stage. Current work in our lab aims to leverage and improve this technology to understand how the lineage controls fate specification. Using a new resonance-scanning confocal microscope, we can collect images with improved axial resolution (0.5 micron) and higher throughput (4-5 embryos at once) while maintaining normal development. By combining the improved images with improved image analysis methods, we can trace the lineage of C. elegans embryos to ~600 cells with about 8 hours of manual curation. We are using a collection of wild type cell lineages to quantitatively analyze normal variability in cell division timing and orientation and cell migration at later stages of embryogenesis, allowing us to more comprehensively answer the question "how invariant is the cell lineage of C. elegans?" We are using these improved methods to understand how gene expression is regulated across the lineage during development. The existing collection of gene expression data shows different spatial and temporal patterns for transcription factors (TFs). We are testing regulatory models within individual cell lineages by using RNAi to perturbing TFs expressed early and measuring changes in potential targets expressed later in the same lineage. We have tested this approach with pal-1, a known regulator of C lineage fate; for some genes, RNAi depletion of pal-1 results in loss of C lineage expression as expected, while for others it results in altered patterns of C lineage expression rather than loss. We are currently expanding this approach to candidate novel lineage regulators and also to understand how developmental decisions are limited to the appropriate time. Regulatory network inference would be more powerful if we knew how all genes' expression varied across the lineage. Genome-wide analysis of FACS sorted cells allows analysis of particular cells or cell types, but fluorescent reporters uniquely and specifically expressed in each of the 558 embryonic cells do not exist, and the cost of sorting and analyzing each of the 558 cells individually would be substantial. We have generated and tested a theoretical framework for deconvoluting the full lineage expression pattern from a smaller set of FACS experiments on 30 reporters chosen to maximize information content. Simulations suggest that this approach could generate high-resolution and accurate measurements of all genes' expression across the full lineage.

Chan, Wesley et al. (2021) International Worm Meeting "BAR-1/beta-catenin and PRY-1/Axin show asymmetric and complementary expression in neuroblasts during C. elegans ventral nerve cord assembly"

Colavita, Antonio, Murray, John, Roenspies, Tony, Evans, Justin, Preston, Elicia, Chan, Wesley

[International Worm Meeting, 2021]

During embryogenesis DD, DA, and DB neuroblasts arise from left and right lineages, move towards the midline and intercalate into a single tract to form the ventral nerve cord (VNC). VNC assembly involves rosette-mediated convergent extension and planar cell polarity (PCP) and Robo pathways [1]. Disruption of this process results in the mispositioning, usually manifested as an anterior displacement, of embryonically-derived motor neurons in the VNC. A more recent analysis of canonical Wnt pathway components also revealed motor neuron position defects. The six DD neurons are evenly spaced along the anterior-posterior (AP) axis and therefore a convenient marker of VNC assembly. We found that mutations in bar-1/beta-catenin and pop-1/TCF display DD spacing defects. For example, in bar-1 and pop-1 mutants, DD2 is displaced anteriorly and therefore in closer proximity to DD1. In contrast, disruption of the negative Wnt signaling regulator PRY-1/Axin results in the posterior displacement of DD1 such that it is in closer proximity to DD2. To begin to understand the roles of BAR-1 and PRY-1, we examined where endogenously-tagged proteins were expressed during VNC assembly. Using CRISPR/Cas9, we generated bar-1(zy97[GFP::bar-1]) and found that BAR-1 is expressed in right but not left side-derived VNC neuroblasts. In contrast, PRY-1::mNG (cp383) is expressed in left but not right side-derived neuroblasts. In pry-1 mutants, BAR-1 is expressed symmetrically in right and left neuroblasts indicating a left-sided repression of BAR-1 signaling. We are examining how neuroblasts intercalate into a single VNC tract in bar-1 and pry-1 mutants to understand the role of this asymmetric expression. Preliminary observations suggest that motor neuron positioning defects begin to manifest around the 1.5-fold stage and later. We hypothesize that BAR-1 asymmetry in right side neuroblasts may help contribute to the proper ordering of DD, DA and DB neurons along the VNC. [1] Shah et al. Dev Cell 2017.

Zacharias, Amanda L. et al. (2013) International Worm Meeting "Regulation of lineage-specific transcription factors by Wnt signaling in C. elegans embryogenesis: more than one way to regulate expression."

Burdick, Joshua T., Preston, Elicia, Walton, Travis, Zacharias, Amanda L., Murray, John I.

[International Worm Meeting, 2013]

The invariant C. elegans lineage robustly generates diverse cell fates, with Wnt and other signals regulating expression of lineage-specific transcription factors (TFs). We previously used lineage tracing to map spatiotemporal expression of reporters for 127 embryonic TFs. This identified many "lineally repetitive" (LR) TFs expressed in multiple posterior branches of the lineage, suggesting they are potential targets of Wnt signaling and that they may act combinatorially to specify cell fates. We tested whether 15 of these posterior LR genes, ceh-6, ceh-13, ceh-27, ceh-36, elt-6, mir-57, nhr-25, nhr-67, nob-1, pal-1, pax-3, tlp-1, tbx-11, unc-130, and vab-7, are regulated by Wnt signaling by analyzing expression patterns in embryos treated with RNAi against the Wnt effector TF, pop-1, or its export factor, lit-1. We found all 15 genes had altered expression patterns indicating they are regulated by Wnt signaling. All of the genes' regulatory regions harbor candidate POP-1 binding sites suggesting they could be direct targets, which we are working to test. While previously identified targets are both repressed by POP-1 in unsignaled anterior cells and activated by POP-1 in Wnt-targeted posterior cells, we instead observed two other types of responses. Some genes required POP-1 function only for repression in anterior cells, while others required POP-1 only for activation in posterior cells. To explore the importance of these patterns, we tested the requirement of LR TFs by lineage tracing of mutant embryos, which identified many incorrectly specified cells based on aberrant position, division timing, death, or survival. We identified target genes for LR TFs by RNA-seq of embryos globally overexpressing TFs under a heat-shock promoter and flow-sorted embryonic cells expressing TF reporters. We also used RNA-seq of pop-1 or lit-1 RNAi embryos to identify new potential Wnt targets, some of which are new posterior LR TFs. Together, our results support a general mechanism by which Wnt regulates expression of LR TFs that then combine to ensure correct terminal fates across the lineage.

Zacharias, Amanda L. et al. (2017) International Worm Meeting "The C. elegans anterior Hox gene, ceh-13, activates posterior Hox genes, nob-1 and php-3, to ensure robust posterior cell development."

MURRAY, JOHN ISAAC, Patel, Shaili D., Lavon, Teddy D., Preston, Elicia, Zacharias, Amanda L.

[International Worm Meeting, 2017]

The C. elegans Hox cluster contains only six genes, which are orthologous to only four of the Drosophila Hox transcription factors, possibly because the worm lacks a segmented body plan. Unlike genes in other animal Hox clusters, these Hox genes are not expressed in the same order, anterior to posterior, as their order on the chromosome, suggesting that their regulatory mechanisms may have diverged as well. Furthermore, expression of the anterior Hox ortholog, ceh-13, extends to parts of the extreme posterior of the animal, raising the question of whether it regulates posterior fates. We investigated the regulation and function of three C. elegans Hox genes: ceh-13, an anterior Hox1/labial homolog, and nob-1 and php-3, two posterior Hox13/Abdominal-B homologs with redundant functions. In most species, expression of these Hox genes does not overlap but we identified high overlap between rescuing nob-1 and ceh-13 transgenes, with ceh-13 expressed one cell cycle earlier than nob-1 in several posterior daughter lineages within the ABp and E lineages. We identified enhancers sufficient to drive part or all of the spatio-temporal expression pattern of both ceh-13 and nob-1, including two putative shadow enhancers. We tested whether ceh-13 might be an upstream regulator of nob-1 by assaying nob-1 reporter expression in ceh-13 mutants. We found that ceh-13 is required for nob-1 reporter expression in the ABplpp and ABprpp sublineages. Arrested ceh-13 mutant embryos and L1 larvae have major defects in head morphology as well as occasional defects in tail morphology similar to those observed in nob-1/php-3 mutants. We tested for similarities in single-cell developmental phenotypes of these two mutants by automated lineage tracing. We found that nob-1/php-3 mutant embryos have widespread defects in cell division and migration in the posterior of the embryo, including non-cell autonomous migration defects, consistent with previous reports. ceh-13 mutants have a fewer defects, most of which are in CEH-13 expressing cells, including in posterior cells where nob-1 expression requires ceh-13. Taken together, these results have several interesting implications. First, it suggests that along with input from the Wnt pathway through POP-1/TCF and SYS-1/ beta -catenin, nob-1 is regulated by different combinations of transcription factors in different sublineages, with only some dependent on ceh-13. Second, we observed that an anterior Hox gene positively regulates expression of a posterior Hox gene and plays an important role in the development of extreme posterior structures. This is consistent with the regulation and function of the Hox genes in C. elegans having diverged from the strict positional roles observed in other animals, suggesting that the evolutionary "rules" for animal body plans may be more flexible than previously appreciated.

Zhao, Zhongying et al. (2009) International Worm Meeting "Automatic Gene Expression Profiling at Single Cell Resolution in both C. elegans and C. briggsae."

Waterston, Robert, Moerman, Don, Vafeados, Dionne, Boeck, Max, Boyle, Thomas, Sandel, Matthew, Murray, John, Flibotte, Stephane, Zhao, Zhongying, Bao, Zhirong, Preston, Elicia, Blick, Dan

[International Worm Meeting, 2009]

We have developed technology that allows automatic determination of the expression profile of fluorescent reporter genes in C. elegans embryos through time with single-cell resolution. The technology is routinely used to trace the expression patterns through the 350-cell stage. Recent advances extend its tracing power to 450 cells and beyond and allow characterization of either GFP or mCherry reporters. We have applied the technology to promoter fusions reporters for more than 100 genes, most of which encode transcription factors differentially expressed in the embryo. We have also analyzed protein-fusion reporters expressed from fosmid transgenes for more than 25 genes, allowing the characterization of posttranscriptional regulation. We will present examples of interesting patterns, including some comparing promoter and protein fusions. We have extended the technology to work in C. briggsae with appropriate strains to allow both GFP and mCherry reporters to be used with results comparable to those obtained in C. elegans. The technology could be readily adapted to the newly identified sister species of C. briggsae, allowing detailed investigation of the effect of the differing degrees of sequence variation on gene expression. We will present comparisons of expression patterns of the same constructs in C. briggsae and C. elegans. To improve the utility of C. briggsae in comparative studies of gene function, we have extended several standard C. elegans methods into C. briggsae. We built a frozen EMS mutagenized library for knock-out deletion screening and utilized it to isolate a cbr-unc-119 deletion allele, which works efficiently as a transgenic selection marker for ballistic bombardment. To facilitate forward mutagenesis studies, we developed a customized SNP-based oligo-array CGH chip for rapid and inexpensive mapping of unknown mutants, using bulk segregant analysis from AF16/HK104 hybrids. We have used the system to map a series of both known and unknown mutants on all six chromosomes. Using these mapping results, we have been able to identify candidate genes in the interval with similar phenotypes in C. elegans and test them through injection-rescue. These tools extend many of the experimental advantages of C. elegans to C. briggsae, which will facilitate future comparative studies.

Liu, Xiao et al. (2009) International Worm Meeting "Analysis of gene regulation and cell fate from single-cell gene expression profiles in C. elegans."

Liu, Xiao, Mericle, Barbara, Kim, Stuart, Aerni, Sarah, Peng, Hanchuan, Myers, Eugene, Preston, Elicia, Jiang, Min, Sanchez-Blanco, Adolfo, Murray, John, Long, Fuhui, Batzoglou, Serafim

[International Worm Meeting, 2009]

To understand gene regulatory networks in development, we developed an experimental pipeline to create a gene expression dataset using images of wCherry-expressing worms as a proof-of-principle that demonstrates important new biological insights can be extracted from single cell gene expression data. Each of the steps in the pipeline can be scaled up, enabling one to generate data much faster in the future. The expression database currently contains images from L1 stage larvae of 93 reporter genes, including 61 transcription factors. We generated their expression profiles in 363 specific cells, and were able to quantitatively analyze expression of each gene as well as the molecular expression signature for each cell. We analyzed the pattern of expression of every gene to determine the relative effect of cell fate and cell lineage. Expression of most genes was strongly correlated with cell fate. However, some cells with identical fate expressed different sets of genes based on their lineage. The most striking examples are nuclei of syncytium hypodermal 7. Twelve hyp7 nuclei are derived from the C lineage and eleven are derived from the AB lineage. The molecular signature for nuclei derived from the C lineage shows a significant difference from that of nuclei derived from the AB lineage. Thus, nuclei in the same syncytial cell can show large differences in gene expression pattern, indicating that there is different transcriptional control in different nuclei. The level of expression of the 93 reporter genes in each cell is a molecular signature for that cell, and can be used as a quantitative measure to determine whether cells have different or related cell fates. We created a developmental activity map based solely on molecular signatures, in which we identify regions of the cell lineage where developmental fates begin to diverge. For each gene, we used the cell lineage and the observed expression levels at the L1 stage to predict when that gene became committed to be expressed in the embryonic lineage. We then searched for embryonic cell divisions in which daughter cells become committed to express a different battery of genes, thereby identifying cell divisions that are asymmetric and revealing when developmental potentials begin to diverge in the embryonic lineage.