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

Walton, Travis, Murray, John

[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"

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

[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, Zhirong Bao, Robert H Waterston

[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.

Plenefisch JD et al. (1994) Worm Breeder's Gazette "Cloning mua-3: Some Observations on the New Molecular Era"

Hedgecock EM, Plenefisch JD

[Worm Breeder's Gazette, 1994]

Cloning mua-3: some observations on the new Molecular Era John Plenefisch and Edward Hedgecock, Dept. of Biology, Johns Hopkins University, Baltimore MD 21218

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

Murray, John, Walton, Travis

[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"

Murray, John, Peng, Felicia

[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.

Parham C et al. (1994) Worm Breeder's Gazette "Tc4 and Tc5: What Makes Them Move and Why It Matters"

Collins JJ, Butze K, Parham C, Beinhorn J

[Worm Breeder's Gazette, 1994]

Tc4 and Tc5: what makes them move and why it matters Christi Parham, Kristie Butze, Joanna Beinhorn and John Collins. Dept. of Biochemistry and Molecular Biology, University of New Hampshire. Durham, NH 03824

Almeida CA et al. (1994) Worm Breeder's Gazette "Function of a Domain of the Myosin Heavy Chain Implicated in Familial Hypertrophic Cardiomyopathy"

Swift KE, Almeida CA, Collins JJ

[Worm Breeder's Gazette, 1994]

Function of a Domain of the Myosin Heavy Chain Implicated in Familial Hypertrophic Cardiomyopathy Craig A. Almeida, Kerry E. Swift and John J. Collins Department of Biochemistry and Molecular, University of New Hampshire, Durham, NH 03820

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.

Chalfie M (1993) J Neurobiol "Touch receptor development and function in Caenorhabditis elegans."

Chalfie M

[J Neurobiol, 1993]

Mutations causing a touch-insensitive phenotype in the nematode Caenorhabditis elegans have been the basis of studies on the specification of neuronal cell fate, inherited neurodegeneration, and the molecular nature of mechanosensory transduction. (C) 1993 John Wiley & sons, Inc.