A genome-scale resource for in vivo tag-based protein function exploration in C. elegans.

Understanding the in vivo dynamics of protein localization and their physical interactions is important for many problems in biology. To enable systematic protein function interrogation in a multicellular context, we built a genome-scale transgenic platform for in vivo expression of fluorescent- and affinity-tagged proteins in Caenorhabditis elegans under endogenous cis regulatory control. The platform combines computer-assisted transgene design, massively parallel DNA engineering, and next-generation sequencing to generate a resource of 14,637 genomic DNA transgenes, which covers 73% of the proteome. The multipurpose tag used allows any protein of interest to be localized in vivo or affinity purified using standard tag-based assays. We illustrate the utility of the resource by systematic chromatin immunopurification and automated 4D imaging, which produced detailed DNA binding and cell/tissue distribution maps for key transcription factor proteins.

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.

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.

Analysis of cell fate from single-cell gene expression profiles in C. elegans.

The C. elegans cell lineage provides a unique opportunity to look at how cell lineage affects patterns of gene expression. We developed an automatic cell lineage analyzer that converts high-resolution images of worms into a data table showing fluorescence expression with single-cell resolution. We generated expression profiles of 93 genes in 363 specific cells from L1 stage larvae and found that cells with identical fates can be formed by different gene regulatory pathways. Molecular signatures identified repeating cell fate modules within the cell lineage and enabled the generation of a molecular differentiation map that reveals points in the cell lineage when developmental fates of daughter cells begin to diverge. These results demonstrate insights that become possible using computational approaches to analyze quantitative expression from many genes in parallel using a digital gene expression atlas.

pop-1/TCF, ref-2/ZIC and T-box factors regulate the development of anterior cells in the C. elegans embryo.

Patterning of the anterior-posterior axis is fundamental to animal development. The Wnt pathway plays a major role in this process by activating the expression of posterior genes in animals from worms to humans. This observation raises the question of whether the Wnt pathway or other regulators control the expression of the many anterior-expressed genes. We found that the expression of five anterior-specific genes in Caenorhabditis elegans embryos depends on the Wnt pathway effectors pop-1/TCF and sys-1/ß-catenin. We focused further on one of these anterior genes, ref-2/ZIC, a conserved transcription factor expressed in multiple anterior lineages. Live imaging of ref-2 mutant embryos identified defects in cell division timing and position in anterior lineages. Cis-regulatory dissection identified three ref-2 transcriptional enhancers, one of which is necessary and sufficient for anterior-specific expression. This enhancer is activated by the T-box transcription factors TBX-37 and TBX-38, and surprisingly, concatemerized TBX-37/38 binding sites are sufficient to drive anterior-biased expression alone, despite the broad expression of TBX-37 and TBX-38. Taken together, our results highlight the diverse mechanisms used to regulate anterior expression patterns in the embryo.

Quantitative Differences in Nuclear ß-catenin and TCF Pattern Embryonic Cells in C. elegans.

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 ß-catenin contribute to context-specific regulation. We investigated this question in Caenorhabditis elegans embryos by quantifying nuclear localization of fluorescently tagged SYS-1/ß-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/ß-catenin activating complex. Specifically, ß-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.

The Bicoid class homeodomain factors ceh-36/OTX and unc-30/PITX cooperate in C. elegans embryonic progenitor cells to regulate robust development.

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.

Overlapping cell population expression profiling and regulatory inference in C. elegans.

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.

Multidimensional regulation of gene expression in the C. elegans embryo.

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.

Diverse transcription factor binding features revealed by genome-wide ChIP-seq in C. elegans.

Regulation of gene expression by sequence-specific transcription factors is central to developmental programs and depends on the binding of transcription factors with target sites in the genome. To date, most such analyses in Caenorhabditis elegans have focused on the interactions between a single transcription factor with one or a few select target genes. As part of the modENCODE Consortium, we have used chromatin immunoprecipitation coupled with high-throughput DNA sequencing (ChIP-seq) to determine the genome-wide binding sites of 22 transcription factors (ALR-1, BLMP-1, CEH-14, CEH-30, EGL-27, EGL-5, ELT-3, EOR-1, GEI-11, HLH-1, LIN-11, LIN-13, LIN-15B, LIN-39, MAB-5, MDL-1, MEP-1, PES-1, PHA-4, PQM-1, SKN-1, and UNC-130) at diverse developmental stages. For each factor we determined candidate gene targets, both coding and non-coding. The typical binding sites of almost all factors are within a few hundred nucleotides of the transcript start site. Most factors target a mixture of coding and non-coding target genes, although one factor preferentially binds to non-coding RNA genes. We built a regulatory network among the 22 factors to determine their functional relationships to each other and found that some factors appear to act preferentially as regulators and others as target genes. Examination of the binding targets of three related HOX factors--LIN-39, MAB-5, and EGL-5--indicates that these factors regulate genes involved in cellular migration, neuronal function, and vulval differentiation, consistent with their known roles in these developmental processes. Ultimately, the comprehensive mapping of transcription factor binding sites will identify features of transcriptional networks that regulate C. elegans developmental processes.

Genome-wide identification of binding sites defines distinct functions for Caenorhabditis elegans PHA-4/FOXA in development and environmental response.

Transcription factors are key components of regulatory networks that control development, as well as the response to environmental stimuli. We have established an experimental pipeline in Caenorhabditis elegans that permits global identification of the binding sites for transcription factors using chromatin immunoprecipitation and deep sequencing. We describe and validate this strategy, and apply it to the transcription factor PHA-4, which plays critical roles in organ development and other cellular processes. We identified thousands of binding sites for PHA-4 during formation of the embryonic pharynx, and also found a role for this factor during the starvation response. Many binding sites were found to shift dramatically between embryos and starved larvae, from developmentally regulated genes to genes involved in metabolism. These results indicate distinct roles for this regulator in two different biological processes and demonstrate the versatility of transcription factors in mediating diverse biological roles.

ngn-1/neurogenin Activates Transcription of Multiple Terminal Selector Transcription Factors in the Caenorhabditis elegans Nervous System.

Proper nervous system development is required for an organism's survival and function. Defects in neurogenesis have been linked to neurodevelopmental disorders such as schizophrenia and autism. Understanding the gene regulatory networks that orchestrate neural development, specifically cascades of proneural transcription factors, can better elucidate which genes are most important during early neurogenesis. Neurogenins are a family of deeply conserved factors shown to be both necessary and sufficient for the development of neural subtypes. However, the immediate downstream targets of neurogenin are not well characterized. The objective of this study was to further elucidate the role of ngn-1/neurogenin in nervous system development and to identify its downstream transcriptional targets, using the nematode Caenorhabditis elegans as a model for this work. We found that ngn-1 is required for axon outgrowth, nerve ring architecture, and neuronal cell fate specification. We also showed that ngn-1 may have roles in neuroblast migration and epithelial integrity during embryonic development. Using RNA sequencing and comparative transcriptome analysis, we identified eight transcription factors (hlh-34/NPAS1, unc-42/PROP1, ceh-17/PHOX2A, lim-4/LHX6, fax-1/NR2E3, lin-11/LHX1, tlp-1/ZNF503, and nhr-23/RORB) whose transcription is activated, either directly or indirectly, by ngn-1 Our results show that ngn-1 has a role in transcribing known terminal regulators that establish and maintain cell fate of differentiated neural subtypes and confirms that ngn-1 functions as a proneural transcription factor in C. elegans neurogenesis.

A lineage-resolved molecular atlas of C. elegans embryogenesis at single-cell resolution.

Caenorhabditis elegans is an animal with few cells but a wide diversity of cell types. In this study, we characterize the molecular basis for their specification by profiling the transcriptomes of 86,024 single embryonic cells. We identify 502 terminal and preterminal cell types, mapping most single-cell transcriptomes to their exact position in C. elegans' invariant lineage. Using these annotations, we find that (i) the correlation between a cell's lineage and its transcriptome increases from middle to late gastrulation, then falls substantially as cells in the nervous system and pharynx adopt their terminal fates; (ii) multilineage priming contributes to the differentiation of sister cells at dozens of lineage branches; and (iii) most distinct lineages that produce the same anatomical cell type converge to a homogenous transcriptomic state.