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1.  Overlapping cell population expression profiling and regulatory inference in C. elegans 
BMC Genomics  2016;17:159.
Background
Understanding gene expression across the diverse metazoan cell types during development is critical to understanding their function and regulation. However, most cell types have not been assayed for expression genome-wide.
Results
We applied a novel approach we term “Profiling of Overlapping Populations of cells (POP-Seq)” to assay differential expression across all embryonic cells in the nematode Caenorhabditis elegans. In this approach, we use RNA-seq to define the transcriptome of diverse partially overlapping FACS-sorted cell populations. This identified thousands of transcripts differentially expressed across embryonic cells. Hierarchical clustering analysis identified over 100 sets of coexpressed genes corresponding to distinct patterns of cell type specific expression. We identified thousands of candidate regulators of these clusters based on enrichment of transcription factor motifs and experimentally determined binding sites.
Conclusions
Our analysis provides new insight into embryonic gene regulation, and provides a resource for improving our knowledge of tissue-specific expression and its regulation throughout C. elegans development.
Electronic supplementary material
The online version of this article (doi:10.1186/s12864-016-2482-z) contains supplementary material, which is available to authorized users.
doi:10.1186/s12864-016-2482-z
PMCID: PMC4772325  PMID: 26926147
C. elegans; Embryonic development; Tissue-specific expression
2.  Quantitative Differences in Nuclear β-catenin and TCF Pattern Embryonic Cells in C. elegans  
PLoS Genetics  2015;11(10):e1005585.
The Wnt signaling pathway plays a conserved role during animal development in transcriptional regulation of distinct targets in different developmental contexts but it remains unclear whether quantitative differences in the nuclear localization of effector proteins TCF and β-catenin contribute to context-specific regulation. We investigated this question in Caenorhabditis elegans embryos by quantifying nuclear localization of fluorescently tagged SYS-1/β-catenin and POP-1/TCF and expression of Wnt ligands at cellular resolution by time-lapse microscopy and automated lineage tracing. We identified reproducible, quantitative differences that generate a subset of Wnt-signaled cells with a significantly higher nuclear concentration of the TCF/β-catenin activating complex. Specifically, β-catenin and TCF are preferentially enriched in nuclei of daughter cells whose parents also had high nuclear levels of that protein, a pattern that could influence developmental gene expression. Consistent with this, we found that expression of synthetic reporters of POP-1-dependent activation is biased towards cells that had high nuclear SYS-1 in consecutive divisions. We identified new genes whose embryonic expression patterns depend on pop-1. Most of these require POP-1 for either transcriptional activation or repression, and targets requiring POP-1 for activation are more likely to be expressed in the cells with high nuclear SYS-1 in consecutive divisions than those requiring POP-1 for repression. Taken together, these results indicate that SYS-1 and POP-1 levels are influenced by the parent cell’s SYS-1/POP-1 levels and this may provide an additional mechanism by which POP-1 regulates distinct targets in different developmental contexts.
Author Summary
The Wnt signaling pathway is active during the development of all multi-cellular animals and also improperly re-activated in many cancers. Here, we use time-lapse microscopy to quantify the nuclear localization of several proteins in response to Wnt signaling throughout early embryonic development in the nematode worm, C. elegans. We find that cells that received a Wnt signal in the previous division respond more strongly to a Wnt signal in the next division, in part by localizing more of the regulator β-catenin to the nucleus. This causes the relative enrichment of Wnt pathway proteins in the nuclei of repeatedly signaled cells, which we show likely impacts the activation of Wnt target genes. This represents a novel mechanism for the regulation of Wnt pathway targets in development and disease.
doi:10.1371/journal.pgen.1005585
PMCID: PMC4619327  PMID: 26488501
3.  The Bicoid Class Homeodomain Factors ceh-36/OTX and unc-30/PITX Cooperate in C. elegans Embryonic Progenitor Cells to Regulate Robust Development 
PLoS Genetics  2015;11(3):e1005003.
While many transcriptional regulators of pluripotent and terminally differentiated states have been identified, regulation of intermediate progenitor states is less well understood. Previous high throughput cellular resolution expression studies identified dozens of transcription factors with lineage-specific expression patterns in C. elegans embryos that could regulate progenitor identity. In this study we identified a broad embryonic role for the C. elegans OTX transcription factor ceh-36, which was previously shown to be required for the terminal specification of four neurons. ceh-36 is expressed in progenitors of over 30% of embryonic cells, yet is not required for embryonic viability. Quantitative phenotyping by computational analysis of time-lapse movies of ceh-36 mutant embryos identified cell cycle or cell migration defects in over 100 of these cells, but most defects were low-penetrance, suggesting redundancy. Expression of ceh-36 partially overlaps with that of the PITX transcription factor unc-30. unc-30 single mutants are viable but loss of both ceh-36 and unc-30 causes 100% lethality, and double mutants have significantly higher frequencies of cellular developmental defects in the cells where their expression normally overlaps. These factors are also required for robust expression of the downstream developmental regulator mls-2/HMX. This work provides the first example of genetic redundancy between the related yet evolutionarily distant OTX and PITX families of bicoid class homeodomain factors and demonstrates the power of quantitative developmental phenotyping in C. elegans to identify developmental regulators acting in progenitor cells.
Author Summary
Animals develop as one initial cell, the fertilized egg, repeatedly divides and its progeny differentiate, ultimately producing diverse cell types. This occurs in large part by the expression of unique combinations of regulatory genes, such as transcription factors, in precursors of each cell type. These early factors are typically reused in precursors of different cell types. The nematode worm Caenorhabditis elegans is a powerful system in which to identify developmental regulators because it has a rapid and reproducible development, yet it shares most of its developmental regulators with more complex organisms such as humans. We used state-of-the-art microscopy and computer-aided cell tracking methods to identify the developmental role of worm homologs of the OTX and PITX genes, whose human homologs play a role in the development of the brain, eye, and pituitary among other tissues. We identified broad roles for OTX in regulating development for many distinct cell types including muscles, neurons and skin, and found a redundant role for both OTX and PITX in a subset of cells. Future studies of these genes should address whether these genes also act redundantly in mammals.
doi:10.1371/journal.pgen.1005003
PMCID: PMC4349592  PMID: 25738873
4.  A quantitative model of normal C. elegans embryogenesis and its disruption after stress 
Developmental biology  2012;374(1):12-23.
The invariant lineage of Caenorhabditis elegans has powerful potential for quantifying developmental variability in normal and stressed embryos. Previous studies of division timing by automated lineage tracing suggested that variability in cell cycle timing is low in younger embryos, but manual lineage tracing of specific lineages suggested that variability may increase for later divisions. We developed improved automated lineage tracing methods that allow routine lineage tracing through the last round of embryonic cell divisions and we applied these methods to trace the lineage of 18 wild-type embryos. Cell cycle lengths, division axes and cell positions are remarkably consistent among these embryos at all stages, with only slight increases in variability later in development. The resulting quantitative 4-dimensional model of embryogenesis provides a powerful reference dataset to identify defects in mutants or in embryos that have experienced environmental perturbations. We also traced the lineages of embryos imaged at higher temperatures to quantify the decay in developmental robustness under temperature stress. Developmental variability increases modestly at 25°C compared with 22°C and dramatically at 26°C, and we identify homeotic transformations in a subset of embryos grown at 26°C. The deep lineage tracing methods provide a powerful tool for analysis of normal development, gene expression and mutants and we provide a graphical user interface to allow other researchers to explore the average behavior of arbitrary cells in a reference embryo.
doi:10.1016/j.ydbio.2012.11.034
PMCID: PMC3548946  PMID: 23220655
robustness; microscopy; cell lineage; image analysis; C. elegans; stress
5.  Deconvolution of gene expression from cell populations across the C. elegans lineage 
BMC Bioinformatics  2013;14:204.
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.
doi:10.1186/1471-2105-14-204
PMCID: PMC3704917  PMID: 23800200
6.  Automated analysis of embryonic gene expression with cellular resolution in C. elegans 
Nature methods  2008;5(8):703-709.
We describe a system that permits the automated analysis of reporter gene expression in Caenorhabditis elegans with cellular resolution continuously during embryogenesis and 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 the direct comparison of the expression of different genes' reporters. We have also used the system to monitor perturbations to normal development involving changes both in cell division timing and in cell fate. Systematic application could reveal the gene activity of each cell throughout development.
doi:10.1038/nmeth.1228
PMCID: PMC2553703  PMID: 18587405
7.  Diverse and Specific Gene Expression Responses to Stresses in Cultured Human CellsD⃞ 
Molecular Biology of the Cell  2004;15(5):2361-2374.
We used cDNA microarrays in a systematic study of the gene expression responses of HeLa cells and primary human lung fibroblasts to heat shock, endoplasmic reticulum stress, oxidative stress, and crowding. Hierarchical clustering of the data revealed groups of genes with coherent biological themes, including genes that responded to specific stresses and others that responded to multiple types of stress. Fewer genes increased in expression after multiple stresses than in free-living yeasts, which have a large general stress response program. Most of the genes induced by multiple diverse stresses are involved in cell-cell communication and other processes specific to higher organisms. We found substantial differences between the stress responses of HeLa cells and primary fibroblasts. For example, many genes were induced by oxidative stress and dithiothreitol in fibroblasts but not HeLa cells; conversely, a group of transcription factors, including c-fos and c-jun, were induced by heat shock in HeLa cells but not in fibroblasts. The dataset is freely available for search and download at http://microarray-pubs.stanford.edu/human_stress/Home.shtml.
doi:10.1091/mbc.E03-11-0799
PMCID: PMC404029  PMID: 15004229

Results 1-7 (7)