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.

Murray, John et al. (2010) C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany "A cellular resolution atlas of gene expression dynamics"

Waterston, Robert, Hyman, Anthony A, Murray, John, Kim, Stuart, Sarov, Mihail, Bao, Zhirong

[C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany, 2010]

We recently developed methods to quantitatively measure gene expression dynamics with cellular resolution in C. elegans embryos. We have now used these methods to analyze the embryonic expression of over 150 genes, mostly transcription factors. The resulting cellular resolution atlas of gene expression dynamics is a powerful tool for studying embryonic fate specification. We identified many genes whose expression was correlated with cell fate or, like Hox genes, position within the embryo. Surprisingly, we also found many genes whose expression was not well-correlated with these features but exhibited repetitive periodic lineage patterns. At least one gene in our dataset can be found which distinguishes the two daughters of 65% of the divisions in early embryogenesis. Most cell lineages do not have a single master fate regulator expressed specifically in only that lineage, however they can combinatorially distinguish over 99% of cell pairs in the embryo. To look specifically for potential posttranscriptional regulation, we examined the expression dynamics of protein-GFP translational reporters. These reporters were largely expressed in the same cells as promoter-fusion reporters for the same gene, but often showed much more complicated dynamics. One common pattern was initial expression in a broad set of cells, followed by dynamic loss of expression in a subset of these that led to a more specific later pattern. Both the appearance and disappearance of TF expression was often tightly associated with cell div ision, and several genes were expressed specifically in all cells during a very limited developmental time window. Together, these results emphasize the complex regulation needed for a cell to adopt the correct fate at the correct time during development.

John Isaac Murray et al. (2004) West Coast Worm Meeting "Automating cell lineage determination and single-cell expression analysis in C. elegans embryos"

John Isaac Murray, Robert H Waterston, Zhirong Bao

[West Coast Worm Meeting, 2004]

We are only beginning to understand the full complexity of how an organism's genome directs its development and behavior. Knowledge of what genes are used in each cell at every stage of development would be a significant step toward a comprehensive understanding. We have begun to develop methods exploiting the invariant lineage of the nematode C. elegans to define gene expression patterns throughout development at the single cell level with high temporal resolution. This will be achieved with two parallel reporter systems. We first computationally track each nucleus in 3D movies of developing embryos expressing histone-GFP fusions in each cell, and thus automatically determine the lineage for each cell. Simultaneously, we will detect a second color, reporter expressed under the control of a candidate transcriptional regulatory region. By mapping the resulting temporal and spatial expression patterns onto the embryonic lineage, we will identify the cells expressing the reporter. Developing these methods will require the creation of new techniques and the modification of existing techniques for the generation of new worms strains, live-cell fluorescence imaging and pattern recognition in images. We plan to assess the accuracy of the method through multiple quality control tests. The completed system will be useful for a wide variety of functional genomics applications and with further adaptation could provide an avenue to comprehensive analysis of gene expression in C. elegans.

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(-).

Peng, Felicia et al. (2021) International Worm Meeting "The Role of mRNA Decay in Embryonic Cell Fate Specification"

Peng, Felicia, Murray, John

[International Worm Meeting, 2021]

During development, cells undergo dynamic changes in gene expression that are required for appropriate cell fate specification. Although the regulation of mRNA degradation contributes to developmental gene expression, its influence on these expression patterns is not well understood relative to that of transcriptional regulation. Like transcriptional regulation, however, the regulation of mRNA decay has the potential to be highly complex. Transcript stability is largely regulated by the binding of protein or RNA factors to cis-regulatory elements within the 3' untranslated region (3' UTR) of transcripts. Considering the great diversity of RNA-binding proteins and miRNAs in eukaryotes, along with differential usage of 3' UTR isoforms, a complex mRNA degradation code may contribute to precise gene expression patterns during development. One relatively well-characterized phenomenon of developmentally regulated mRNA decay occurs during early embryogenesis, in which the widespread degradation of maternal mRNAs is required for the control of development to switch from maternally to zygotically encoded products. Increasing evidence is also emerging for the importance of zygotic mRNA degradation in cell fate decisions, though these studies tend to occur on a gene-by-gene basis or in the context of cell culture systems. Thus the extent of developmentally regulated zygotic mRNA decay remains unclear. We hypothesize that the regulation of zygotic mRNA degradation contributes to appropriate cell fate decisions throughout embryogenesis. To explore this, we are examining mRNA decay rates during embryonic development in Caenorhabditis elegans. By sequencing embryonic cells treated with a transcription inhibition time course, we have measured mRNA half-lives globally in C. elegans embryos. We found that half-lives can vary by more than an order of magnitude from gene to gene, and differences in mRNA stability correlate with functional classes of genes. Furthermore, analysis of the half-life distributions of tissue- and developmental stage-enriched genes suggests that mRNA decay is differentially regulated across different tissues and stages. Future work should extend these results to measure differential degradation of the same mRNAs in different cell types and stages. Our results highlight that zygotic mRNA degradation is a highly regulated process with tissue- and stage-specific dynamics during development.

Sivaramakrishnan, Priya et al. (2019) International Worm Meeting "Control of transcription rates in the Caenorhabditis elegans embryo."

MURRAY, JOHN ISAAC, Sivaramakrishnan, Priya

[International Worm Meeting, 2019]

Embryonic cell fate decisions rely on transcription programs that are coordinated in both space and time. Temporal coordination is particularly important in the rapidly dividing C. elegans embryo, where developmental regulators need to be transcribed quickly enough to instruct fate decisions before the next cell cycle. This requires embryonic transcription to be very accurate. But transcription is known to be noisy, occurring in bursts followed by periods of quiescence. Thus, how transcriptional noise is prevented or reduced to allow for developmental robustness is unclear. Several early zygotic regulators in C. elegans are transcribed at very high rates. Analysis of single molecule RNA FISH (smFISH) data shows that the mRNA levels of the endoderm regulator end-3 increase from zero to 600 transcripts in one cell within a 15-minute cell cycle. We hypothesize that high transcription rates of critical embryonic regulators can promote developmental robustness. To determine whether such high rates occur for other genes, we used single-cell RNA sequencing (scRNA-seq) data from 84,625 embryonic cells to measure maximum transcript levels at single cell resolution. We identified 20 candidate genes with high transcript abundance above a set threshold. We are using smFISH to validate the high expression of these genes and to measure the noise associated with their transcription. The expression of one candidate gene, ceh-51, a muscle-specific transcription factor is similar to end-3, producing 600 transcripts per cell. To estimate transcriptional bursting during ceh-51 expression, we targeted smFISH probes to ceh-51 introns to label sites of nascent transcription. Surprisingly, we detect transient accumulation of nuclear intron-containing transcripts within the nucleus outside of transcriptionally active sites. This suggests that transcription and splicing may be decoupled for ceh-51, which we are exploring further. scRNA-seq identifies genes with high transcript abundance in single cells, but does not directly measure transcription rates. Hence, we are combining the metabolic labeling of mRNAs with scRNA-seq to detect recent transcription events and quantify genome-wide transcription rates during embryonic development. Our approach and methodologies can be applied to identify universal mechanisms that support rapid and temporally precise transcription.

Rumley, Jonathan et al. (2015) International Worm Meeting "Enhancer Regulatory Logic Controlling the Muscle-Hypodermal Differentiation Decision in the Caenorhabditis elegans C Lineage."

Zacharias, Amanda, Murray, John, Rumley, Jonathan

[International Worm Meeting, 2015]

During development, tissue-specific transcription factors direct differentiation decisions as undifferentiated cells adopt various fates. Uncovering how enhancers regulate the cell specificity, timing of onset, and duration of maintenance of target gene expression is thus vital for understanding normal development. We have begun to study the enhancers that control the expression of the master regulators of muscle and hypodermal specification in the C. elegans C lineage through the regulation of the terminal selector genes elt-1 and hlh-1.Previous work from other labs showed that PAL-1 is expressed in all C lineage cells, and induces the expression of the hypodermal regulator elt-1 and the muscle regulator hlh-1 as well as other targets. POP-1 inhibits the expression of hlh-1 in the anterior C granddaughters (hypodermal progenitors), and may induce its expression in the posterior granddaughters (muscle progenitors). In contrast, elt-1 is initially expressed in all C descendants but expression is maintained only in the anterior C granddaughters and its maintenance may be repressed by hlh-1 in muscle progenitors. An enhancer (enh1) of hlh-1 has two PAL-1 binding sites but no high affinity POP-1 binding site and drives expression in embryonic blastomeres, consistent with it being responsible for hlh-1 initiation. The cis elements controlling elt-1 initiation in the C lineage, however, are unknown.We plan to study the role of PAL-1 and POP-1 binding sites in regulating enh1-driven expression by incorporating enh1 into an enhancer-reporter construct, changing the number and affinity of PAL-1 and POP-1 binding sites, and inserting modified enh1 enhancer-reporter constructs into the genome. In parallel, we plan to identify elt-1 enhancers bearing PAL-1 and POP-1 binding sites that drive expression in C lineage hypodermal progenitors. We will track lineal reporter expression by time-lapse confocal microscopy and automated lineage tracing. By manipulating the number, affinity and organization of binding sites in these enhancers and in synthetic enhancers, we hope to begin to understand the logic controlling embryonic gene expression.

Rumley, Jonathan et al. (2021) International Worm Meeting "Regulation of anterior genes in the C. elegans embryo"

Rumley, Jonathan, Zacharias, Amanda, Murray, John

[International Worm Meeting, 2021]

Anterior-posterior patterning is vital for embryonic development. In animals from worms to humans, Wnt patterns the anterior-posterior axis. Wnt drives expression of several genes expressed in lineages derived from posterior daughters. Previous work in our lab and others showed that many of these posterior genes depend on Wnt pathway component POP-1/TCF either for posterior expression or anterior repression. There are nearly as many genes expressed in lineages derived from anterior daughter cells as there are posterior genes and much less is known about how these anterior genes are regulated. Anterior genes may be regulated by Wnt indirectly through a canonical Wnt target, directly by Wnt by non-canonical means, or by non-Wnt mechanisms. I have found that several anterior genes depend on POP-1 for expression in an opposite manner to posterior genes. To determine which transcription factors regulate an anterior gene's expression, I dissected the cis-regulation of the anterior gene ref-2/ZIC1. I used enhancer-reporter strains to identify two ref-2 enhancers including one that explains its anterior-specific embryonic expression. I examined the expression patterns driven by enhancer fragments to identify minimal fragments sufficient to drive anterior expression. A fragment of one of these enhancers bears five putative TBX-37/38 binding sites, one of which I found to be essential for anteriorly biased expression. Synthetic enhancer-reporter constructs bearing multiple copies of this t-box site show that it is sufficient to drive anterior expression. By performing RNAi knockdown of pop-1, I have determined that the anterior expression driven by this site is dependent on pop-1. Overall this work suggests that multiple transcription factors play a role in anterior patterning of the C. elegans embryo.

Sivaramakrishnan, Priya et al. (2021) International Worm Meeting "Transcription rates in the early embryo"

Sivaramakrishnan, Priya, MURRAY, JOHN ISAAC, Watkins, Cameron

[International Worm Meeting, 2021]

Rapid changes in gene expression are necessary for cells to respond to environmental, immunological or developmental signals. As a critical first step, high transcription synthesis rates are required during these responses. In the rapidly dividing C. elegans embryo, where cell cycles are as short as 15 minutes, temporal constraints on transcription are particularly severe. Early cell fate specification genes have to be transcribed at very high rates to accumulate to threshold levels within short developmental time windows. Studies in other labs have shown that reducing the normally high expression levels of some endodermal regulators results in aberrant intestinal fate specification. We hypothesize that high rates of transcription could be required for the fate decisions in several additional early cells. We estimated transcription rates in the early embryo by analyzing single cell RNA sequencing data and found that maximal rates vary across cell types, lineages, and embryonic stages. We have validated some of these by single molecule RNA fluorescence in situ hybridization (smFISH) and identified features of rapidly transcribed genes such as short gene length, shorter and fewer introns, and lack of trans-splicing. We found several motifs enriched in the core promoters of high-rate genes including the initiator element (inr), sites for the SP1 transcription factor, and binding sites for lineage-specific regulators. Using the high-rate mesoderm specification gene ceh-51 as a test case, we measured the effect of these promoter elements on transcription rates using CRISPR-mediated promoter mutagenesis and smFISH. Our analysis shows significant redundancies built into the ceh-51 core promoter with modest contributions to rate from each motif, suggesting that control of transcription rates is surprisingly robust. We are currently designing experiments to test for a direct impact of transcription rate on cell fate. Ultimately, we hope to understand how transcription parameters such as bursting are influenced by rate control and how this contributes to gene expression precision underlying the invariant C. elegans embryonic lineage.

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.