Murray JI et al. (2006) Nat Protoc "The lineaging of fluorescently-labeled Caenorhabditis elegans embryos with StarryNite and AceTree."

Bao Z, Murray JI, Boyle TJ, Waterston RH

[Nat Protoc, 2006]

Lineage analysis of Caenorhabditis elegans is a powerful tool for characterizing developmental phenotypes and embryonic gene-expression patterns. We present a detailed protocol for the lineaging of embryos by computational analysis of 4D images of embryos that ubiquitously express histone-GFP (green fluorescent protein) fusion proteins through the 350 cell stage followed by manual editing. We describe how to optimize imaging settings for this purpose, the use of the lineage-extraction software, StarryNite, and the lineage-editing software, AceTree. In addition, we describe a useful polymer bead mounting technique for C. elegans embryos that has several advantages compared with the standard agar pad mounting technique. The protocol requires about 1 h of user time spread over 2 days to generate the raw lineage, and an additional 2 or 4 h to edit the lineage to the 194- or 350-cell stage, respectively.

Murray JI et al. (2012) Genome Res "Multidimensional regulation of gene expression in the C. elegans embryo."

Preston E, Murray JI, Boeck ME, Bao Z, Zhao Z, Vafeados D, Boyle TJ, Waterston R, Weisdepp P, Mericle B

[Genome Res, 2012]

How cells adopt different expression patterns is a fundamental question of developmental biology. We quantitatively measured reporter expression of 127 genes, primarily transcription factors, in every cell and with high temporal resolution in C. elegans embryos. Embryonic cells are highly distinct in their gene expression; expression of the 127 genes studied here can distinguish nearly all pairs of cells, even between cells of the same tissue type. We observed recurrent lineage-regulated expression patterns for many genes in diverse contexts. These patterns are regulated in part by the TCF-LEF transcription factor POP-1. Other genes' reporters exhibited patterns correlated with tissue, position, and left-right asymmetry. Sequential patterns both within tissues and series of sublineages suggest regulatory pathways. Expression patterns often differ between embryonic and larval stages for the same genes, emphasizing the importance of profiling expression in different stages. This work greatly expands the number of genes in each of these categories and provides the first large-scale, digitally based, cellular resolution compendium of gene expression dynamics in live animals. The resulting data sets will be a useful resource for future research.

Murray JI (2018) Wiley Interdiscip Rev Dev Biol "Systems biology of embryonic development: Prospects for a complete understanding of the Caenorhabditis elegans embryo."

Murray JI

[Wiley Interdiscip Rev Dev Biol, 2018]

The convergence of developmental biology and modern genomics tools brings the potential for a comprehensive understanding of developmental systems. This is especially true for the Caenorhabditis elegans embryo because its small size, invariant developmental lineage, and powerful genetic and genomic tools provide the prospect of a cellular resolution understanding of messenger RNA (mRNA) expression and regulation across the organism. We describe here how a systems biology framework might allow large-scale determination of the embryonic regulatory relationships encoded in the C. elegans genome. This framework consists of two broad steps: (a) defining the "parts list"-all genes expressed in all cells at each time during development and (b) iterative steps of computational modeling and refinement of these models by experimental perturbation. Substantial progress has been made towards defining the parts list through imaging methods such as large-scale green fluorescent protein (GFP) reporter analysis. Imaging results are now being augmented by high-resolution transcriptome methods such as single-cell RNA sequencing, and it is likely the complete expression patterns of all genes across the embryo will be known within the next few years. In contrast, the modeling and perturbation experiments performed so far have focused largely on individual cell types or genes, and improved methods will be needed to expand them to the full genome and organism. This emerging comprehensive map of embryonic expression and regulatory function will provide a powerful resource for developmental biologists, and would also allow scientists to ask questions not accessible without a comprehensive picture. This article is categorized under: Invertebrate Organogenesis > Worms Technologies > Analysis of the Transcriptome Gene Expression and Transcriptional Hierarchies > Gene Networks and Genomics.

Murray JI et al. (2012) Cold Spring Harb Protoc "Automated lineage and expression profiling in live Caenorhabditis elegans embryos."

Murray JI, Bao Z

[Cold Spring Harb Protoc, 2012]

Describing gene expression during animal development requires a way to quantitatively measure expression levels with cellular resolution and to describe how expression changes with time. Fluorescent protein reporters make it possible to measure expression dynamics in live cells by time-lapse microscopy, but it can be challenging to identify expressing cells in complex tissues and to compare expression across organisms. This protocol describes how to use automated lineage analysis to identify cells in Caenorhabditis elegans embryos expressing fluorescent reporters and how to quantify that expression with cellular resolution. Because C. elegans develops through an invariant pattern of cell divisions, every cell's identity and future fate can be predicted from its pattern of previous cell divisions. Automated analysis of images collected from embryos expressing a fluorescent histone transgene in all cells allows lineage tracing and cell identification. This provides a scaffold with which to describe expression of a second color reporter such as a fusion of a second fluorescent protein to a gene of interest or its regulatory sequences. These methods can also be used for analysis of reporter expression, cell division timing, and cell position in genetically perturbed embryos. The protocol describes how to prepare C. elegans strains containing nuclear-expressed fluorescent reporters, collect images of appropriate quality from embryos, perform automated lineage analysis, manually edit and curate the lineage, and, finally, extract and display reporter signals.

Murray JI et al. (2008) Nat Methods "Automated analysis of embryonic gene expression with cellular resolution in C. elegans."

Nicholas TJ, Waterston RH, Bao Z, Mericle BL, Zhao Z, Sandel MJ, Murray JI, Boyle TJ, Boeck ME

[Nat Methods, 2008]

We describe a system that permits the automated analysis of reporter gene expression in Caenorhabditis elegans with cellular resolution continuously during embryogenesis. We demonstrate its utility by defining the expression patterns of reporters for several embryonically expressed transcription factors. The invariant cell lineage permits the automated alignment of multiple expression profiles, allowing direct comparison of the expression of different genes'' reporters. We also used this system to monitor perturbations to normal development involving changes both in cell-division timing and in cell fate. Systematic application of this system could reveal the gene activity of each cell throughout development.

Murray JI et al. (2022) PLoS Genet "The anterior Hox gene ceh-13 and elt-1/GATA activate the posterior Hox genes nob-1 and php-3 to specify posterior lineages in the C. elegans embryo."

Preston E, Patel SD, Hsiao E, Bennett BA, Zacharias AL, Anderson BD, Amom P, Peng F, Crawford JP, Murray JI, Rumley JD, Sivaramakrishnan P, Lavon TD

[PLoS Genet, 2022]

Hox transcription factors play a conserved role in specifying positional identity during animal development, with posterior Hox genes typically repressing the expression of more anterior Hox genes. Here, we dissect the regulation of the posterior Hox genes nob-1 and php-3 in the nematode C. elegans. We show that nob-1 and php-3 are co-expressed in gastrulation-stage embryos in cells that previously expressed the anterior Hox gene ceh-13. This expression is controlled by several partially redundant transcriptional enhancers. These enhancers act in a ceh-13-dependant manner, providing a striking example of an anterior Hox gene positively regulating a posterior Hox gene. Several other regulators also act positively through nob-1/php-3 enhancers, including elt-1/GATA, ceh-20/ceh-40/Pbx, unc-62/Meis, pop-1/TCF, ceh-36/Otx, and unc-30/Pitx. We identified defects in both cell position and cell division patterns in ceh-13 and nob-1;php-3 mutants, suggesting that these factors regulate lineage identity in addition to positional identity. Together, our results highlight the complexity and flexibility of Hox gene regulation and function and the ability of developmental transcription factors to regulate different targets in different stages of development.

Sivaramakrishnan P et al. (2019) Elife "Silencing the alternative."

Murray JI, Sivaramakrishnan P

[Elife, 2019]

The transcription factor <i>ztf-11</i> promotes neuronal differentiation by repressing other cell fates in the nematode worm <i>C. elegans</i>.

Bao Z et al. (2011) Cold Spring Harb Protoc "Mounting Caenorhabditis elegans embryos for live imaging of embryogenesis."

Murray JI, Bao Z

[Cold Spring Harb Protoc, 2011]

Caenorhabditis elegans has been a key model organism for biomedical research. Light microscopy has played a central role in C. elegans biology. C. elegans is transparent throughout its life cycle, and its physical size, from 50 m (embryos) to 1 mm (adults), is well suited for light microscopy. Furthermore, it has an invariant body plan that arises from an invariant cell lineage. A wide range of biological processes, from patterns of gene expression to cell migration to neuronal activity, can be readily observed in single cells with a well-defined developmental context. This protocol describes how to collect and mount young C. elegans embryos for live imaging throughout embryogenesis.

Burdick JT et al. (2013) BMC Bioinformatics "Deconvolution of gene expression from cell populations across the C. elegans lineage."

Murray JI, Burdick JT

[BMC Bioinformatics, 2013]

BACKGROUND: Knowledge of when and in which cells each gene is expressed across multicellular organisms is critical in understanding both gene function and regulation of cell type diversity. However, methods for measuring expression typically involve a trade-off between imaging-based methods, which give the precise location of a limited number of genes, and higher throughput methods such as RNA-seq, which include all genes, but are more limited in their resolution to apply to many tissues. We propose an intermediate method, which estimates expression in individual cells, based on high-throughput measurements of expression from multiple overlapping groups of cells. This approach has particular benefits in organisms such as C. elegans where invariant developmental patterns make it possible to define these overlapping populations of cells at single-cell resolution. RESULT: We implement several methods to deconvolve the gene expression in individual cells from population-level data and determine the accuracy of these estimates on simulated data from the C. elegans embryo. CONCLUSION: These simulations suggest that a high-resolution map of expression in the C. elegans embryo may be possible with expression data from as few as 30 cell populations.

Liu J et al. (2023) Genetics "Mechanisms of lineage specification in Caenorhabditis elegans."

Liu J, Murray JI

[Genetics, 2023]

The studies of cell fate and lineage specification are fundamental to our understanding of the development of multicellular organisms. Caenorhabditis elegans has been one of the premiere systems for studying cell fate specification mechanisms at single cell resolution, due to its transparent nature, the invariant cell lineage, and fixed number of somatic cells. We discuss the general themes and regulatory mechanisms that have emerged from these studies, with a focus on somatic lineages and cell fates. We next review the key factors and pathways that regulate the specification of discrete cells and lineages during embryogenesis and postembryonic development; we focus on transcription factors and include numerous lineage diagrams that depict the expression of key factors that specify embryonic founder cells and postembryonic blast cells, and the diverse somatic cell fates they generate. We end by discussing some future perspectives in cell and lineage specification.