Walton, Travis et al. (2013) International Worm Meeting "ceh-36 regulates cell fate patterning during embryogenesis."

Walton, Travis, Murray, John

[International Worm Meeting, 2013]

We previously used lineage tracing analysis to define the expression patterns of transcriptional and translational reporters of over 100 transcription factors (TFs) in embryos through the 350-cell stage. One finding of this work was the unexpected prevalence of "lineally repetitive" TF expression patterns in multiple lineages not related by terminal fate. This suggests that these TFs may mediate the conversion of lineage identity into patterns of cell fate. We have focused on one lineally repetitive TF, the Otx homeodomain factor ceh-36, which is expressed in several lineages that generate 251 cells with diverse cell fates. Although there are three Otx TFs that are functionaly redundant in some cell types, previous genetic studies demonstrated roles for ceh-36 distinct from its paralogs ttx-1 and ceh-37. Therefore, we investigated the requirement of ceh-36 as a regulator of lineage identity during embryogenesis. We tested for defects in cell cycle events and cell position at single-cell resolution by 4D microscopy and lineage tracing of ceh-36(-) mutant embryos through comma stage and comparison with our quantitative model of wildtype embryogenesis. We analyzed two putative null alleles, ceh-36(ok795) or ceh-36(ky646), which are both characterized by partially penetrant larval lethality. Quantitative analysis identified at least 29 cells with defective lineage patterns or positions, which included failed cell death and cell migrations to inappropriate left/right bilateral position. Most defects were partially penetrant, possibly due to redundancy with other regulators. Cells identified by our quantitative analysis have been validated using cell-type specific reporters and indicate defective cell-fate specification. Our work demonstrates that ceh-36 plays a broad role specifying diverse cell-types whose common feature is shared lineage ancestry. Our analysis likely underestimates the number of cells that require ceh-36 function because it did not identify other cells whose identity, but not position or lineage pattern, are known to be defective in ceh-36(-).

Murray, John et al. (2015) International Worm Meeting "Temporal constraints and regulation of embryonic gene expression."

Murray, John, Walton, Travis

[International Worm Meeting, 2015]

Each cell in a developing organism needs to adopt expression patterns and behaviors appropriate not only for its future tissue identity but also to the current developmental time. The need for temporal coordination is especially important in developmental contexts such as the C. elegans embryo, where rapid cell division occurs simultaneously with complex patterns of cell fate decisions, and where temporal misregulation could significantly alter the final pattern of cell fates.One major constraint placed on zygotic expression is the punctuation created by mitosis. Early mitotic cycles can be less than 15 minutes, with potentially less than 10 minutes of interphase; transcription is thought to cease during mitosis. The burden of transcribing and translating regulators of cell fate in rapidly and in sufficient quantities to allow them to act in subsequent divisions is thus substantial. Part of the worm's solution to this problem may be structural. Early zygotic genes are strongly biased towards short primary transcript lengths (and little or no introns) and they often exist in duplicated gene families. Together, these features would be predicted to allow faster production of initial transcripts and substantially higher maximum transcript counts in a short cell cycle.A notable feature of many developmental regulators is the apparent synchronization of their expression with the cell cycle. In many cases, TF proteins accumulate in the nucleus and mRNA transcripts begin to be transcribed immediately after mitosis. Expression of numerous endogenous RNAs remains synchronized with the cell cycle across a range of developmental rates as controlled by temperature or clk-1 mutations. While previous work showed that transcription of several tissue-specific genes does not strictly require mitosis, our data suggest that some form of cell cycle gating may occur. For example, we find that a FKH-4::GFP fusion protein, which normally accumulates transiently in all nuclei immediately following the AB8-AB16 division retains this postmitotic timing when the cell cycle length is perturbed by small amounts. However, larger cell cycle perturbations can shift the onset of FKH-4::GFP accumulation a full cycle earlier, to the AB4-AB8 division. Again, the punctuation of mitosis may provide, by virtue of the lack of transcription, a window where any small asynchronies in developmental processes can be reset. While most of these mechanisms remain unproven, they would help ensure that gene regulation remains synchronized across the diverse developmental conditions experienced by the worms in the wild.

King, Brian et al. (2009) International Worm Meeting "The identification of glo-1(-) suppressors."

Hermann, Greg, Walton, Travis, Scavarda, Emily, King, Brian

[International Worm Meeting, 2009]

Lysosome-related organelles (LROs) comprise a class of cell-type specific compartments with specialized functions. In mammals, defects in their formation result in Hermansky-Pudlak syndrome. The genes controlling the biogenesis of LROs are conserved in metazoa and include subunits of the AP-3 and HOPS complexes and Rab32/38. GLO-1, a homologue of Rab32/38, is localized to and is required for the formation of C. elegans gut granules, LROs containing fat and birefringent material found only within intestinal cells. In an effort to better understand the activity of GLO-1, we have carried out a screen for mutations the suppress gut granule biogenesis defects associated with the loss of glo-1 function. To date, we have molecularly characterized 16 mutant alleles of glo-1: 6 are missense alleles altering amino acid residues conserved in all Rab GTPase, 3 are amber nonsense alleles, 2 are opal nonsense alleles, 2 are alleles predicted to alter splicing, and 2 are alleles that delete the entire glo-1 gene. All of the alleles result in the complete loss of birefringent material from embryonic intestinal cells and its mislocalization into the embryonic intestinal lumen. In order to identify both bypass and allele specific suppressors we screened for suppressors of glo-1(kx8), an allele that changes Asp67 to Asn. In other small GTPases, this residue has been shown to be essential for their function. We carried out a screen of F2 embryos within approximately 100,000 mutagenized glo-1(kx8) F1 adults for the presence of birefringent compartments within their intestinal cells. From the screen we identified 8 glo-1 suppressors (kx101-kx108) that fall into 2 distinct classes. Six of the alleles are recessive and weakly restore birefringent compartments in glo-1(kx8) embryos. However, other markers of gut granules do not appear to be similarly restored, suggesting that these suppressors alter the trafficking or formation of the birefringent material. Two suppressors, kx105 and kx106, dominantly and with high penetrance, suppress the loss and mislocalization of birefringent material. Suppressed embryos contain organelles that exhibit all of the hallmarks of normal gut granules. kx105 suppresses the loss of gut granules in multiple glo-1 alleles, including those containing very early premature stop codons, indicating that it bypasses the function of glo-1 in gut granule biogenesis. We are currently investigating whether the dominant suppressors can similarly suppress the defects in gut granule biogenesis exhibited by AP-3 and HOPS mutants. The dominant suppressors are not present on the X chromosome where glo-1 is located, and we are carrying out assays to identify the gene(s) altered by these alleles.

Osborn, Gregory et al. (2013) International Worm Meeting "Genomic analysis of the duct and pore cells reveals novel effectors and regulators of morphogenesis."

Walton, Travis, Osborn, Gregory, Sundaram, Meera, Murray, John

[International Worm Meeting, 2013]

Organogenesis involves complex cellular processes such as migration, morphological changes and differentiation, which are frequently orchestrated by the integration of regulatory networks. To understand how these cellular behaviors are coordinated at single cell resolution, we are studying the C. elegans excretory system, which contains three tubular cells: the duct, pore and canal cell. During embryogenesis, the excretory duct and pore cell first exhibit mesenchymal characteristics as they migrate from disparate locations to meet the canal cell at the ventral midline, where they change shape, differentiate into epithelia, and convert into a contiguous three cell tubular organ. To identify molecular factors involved in these complex processes, we are characterizing the transcriptomes of these cells during development. To isolate the duct and pore, we have taken a fluorescence-activated cell sorting (FACS) strategy, which allows for isolation of cells expressing a fluorescent reporter of interest. Specific early markers for the duct and pore during organogenesis are not known; thus we have taken an intersectional approach, sorting for cells expressing both ceh-6::GFP and hlh-16p::mCherry in C. elegans embryos. The expression of these reporters overlaps exclusively in the duct and pore, as well as their sisters, the DB1/3 motor neurons. RNA-seq of these cells shows an enrichment of factors known to be important in the formation of the excretory system, such as EGF signaling components (let-23, lin-1) and genes involved in proper tube formation and function (aff-1, let-4, lpr-1). Interestingly, novel factors were also enriched, including many HOX transcription factors and proteins predicted to be secreted or membrane-localized. This dataset could be augmented in the future by using additional available markers to separate the duct/pore from the DB1/3 motor neurons by FACS and determining the expression signatures unique and shared between these cells. This work will provide a framework to identify novel candidates to functionally test as regulators and effectors of the complex cellular behaviors of the developing excretory system.

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.

Somhegyi, Hannah et al. (2011) International Worm Meeting "Investigating the function of the GLO-1 Rab GTPase in lysosome-related organelle biogenesis."

Hermann, Greg J., King, Brian, Walton, Travis, Curtin, Thomas, Somhegyi, Hannah

[International Worm Meeting, 2011]

Lysosome-related organelles (LROs) comprise a class of cell-type restricted compartments with specialized functions. In mammals, they include melanosomes, platelet dense granules, and lamellar bodies; defects in their formation result in Hermansky-Pudlak syndrome. The genes controlling the biogenesis of LROs are conserved in metazoa and include subunits of the AP-3 and HOPS complexes and Rab32/38. Our studies have shown that GLO-1, a homologue of Rab32/38, is required for the formation of C. elegans gut granules, lysosome-related organelles containing autofluorescent and birefringent material found only within intestinal cells. Presently, the function of Rab32/38 proteins in any system remains unknown. In an effort to better understand the role of GLO-1, we have analyzed the activity of 16 different alleles of glo-1. All of the alleles result in the complete loss of birefringent material from embryonic intestinal cells and its mislocalization into the embryonic intestinal lumen. Interestingly, a missense allele glo-1(kx8), which changes an invariant residue in the G3/switch II domain of small GTPases, retains weak activity. We carried out a genetic screen for mutations the suppress the gut granule biogenesis defect associated with glo-1(kx8). We identified two alleles, kx105 and kx106, which map to similar locations, that are semi-dominant, gain-of-function, and allele-specific suppressors of glo-1(kx8). Notably, the gut granules that are restored in glo-1(kx8); kx105 lack some gut granule associated proteins, which are mistargeted to conventional lysosomes. However, the gut granules that are restored utilize previously identified gut granule trafficking pathways. Together, these results indicate that the suppressor restores some but not all GLO-1 activities and that the identification of the suppressor will provide insights into the function of the protein. Rabs act via interactions with effector molecules when they are in the GTP bound state. We have found that a GTP-locked and functional form of GLO-1 is present on organelles that are distinct from mature gut granules. They do not appear to be early or late endosomes and are thus divergent from organelles participating in trafficking to conventional lysosomes. These compartments are the likely site of GLO-1 activity and identify intermediates in gut granule trafficking.

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.

Zacharias, Amanda L. et al. (2013) Worm Breeder's Gazette "Visualization of embryonic development with a downloadable 4D quantitative model"

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

[Worm Breeder's Gazette, 2013]

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.

Shi, Cheng et al. (2019) International Worm Meeting "Insulin-like peptides and the mTOR-TFEB pathway protect C. elegans hermaphrodites from Mating-induced Death."

Shi, Cheng, Murphy, Coleen

[International Worm Meeting, 2019]

C. elegans lifespan is shortened by mating, but must be delayed long enough for successful reproduction. Susceptibility to brief mating-induced death increases with age; surprisingly, this is not due to declining health, but to loss of protection upon self-sperm depletion. Self-sperm maintains expression of a DAF-2 insulin-like antagonist, INS-37, which promotes the nuclear localization of HLH-30/TFEB, a pro-longevity regulator. Mating induces the agonist INS-8, promoting HLH-30 nuclear exit and subsequent death. In opposition to the protective role of HLH-30 and DAF-16/FOXO, TOR/LET-363 and the IIS-regulated Zn-finger transcription factor PQM-1 promote seminal-fluid-induced killing. Self-sperm maintenance of nuclear HLH-30/TFEB allows hermaphrodites to resist mating-induced death, increasing the chances that mothers will survive through reproduction. The hijacking of the IIS pathway by males is combated by the mother's expression of an insulin antagonist that keeps her healthy through the activity of pro-longevity factors, as long as she has her own sperm to utilize. ** If possible, I would prefer that this abstract please be considered with the abstract submitted by Lauren N. Booth, Travis J. Maures, Robin W. Yeo, and Anne Brunet ("Self-sperm induce resistance to the detrimental effects of sexual encounters with males in hermaphroditic nematodes").