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1.
Cell ; 150(4): 855-66, 2012 Aug 17.
Article in English | MEDLINE | ID: mdl-22901814

ABSTRACT

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.


Subject(s)
Animals, Genetically Modified , Caenorhabditis elegans Proteins/analysis , Caenorhabditis elegans/genetics , Genetic Engineering/methods , Genome, Helminth , Transcription Factors/analysis , Animals , Caenorhabditis elegans/chemistry , Caenorhabditis elegans/metabolism , Caenorhabditis elegans Proteins/genetics , Transcription Factors/genetics
2.
PLoS Genet ; 19(9): e1010944, 2023 09.
Article in English | MEDLINE | ID: mdl-37721936

ABSTRACT

Some types of collagens, including transmembrane MACIT collagens and C. elegans cuticle collagens, are N-terminally cleaved at a dibasic site that resembles the consensus for furin or other proprotein convertases of the subtilisin/kexin (PCSK) family. Such cleavage may release transmembrane collagens from the plasma membrane and affect extracellular matrix assembly or structure. However, the functional consequences of such cleavage are unclear and evidence for the role of specific PCSKs is lacking. Here, we used endogenous collagen fusions to fluorescent proteins to visualize the secretion and assembly of the first collagen-based cuticle in C. elegans and then tested the role of the PCSK BLI-4 in these processes. Unexpectedly, we found that cuticle collagens SQT-3 and DPY-17 are secreted into the extraembryonic space several hours before cuticle matrix assembly. Furthermore, this early secretion depends on BLI-4/PCSK; in bli-4 and cleavage-site mutants, SQT-3 and DPY-17 are not efficiently secreted and instead form large intracellular puncta. Their later assembly into cuticle matrix is reduced but not entirely blocked. These data reveal a role for collagen N-terminal processing in intracellular trafficking and the control of matrix assembly in vivo. Our observations also prompt a revision of the classic model for C. elegans cuticle matrix assembly and the pre-cuticle-to-cuticle transition, suggesting that cuticle layer assembly proceeds via a series of regulated steps and not simply by sequential secretion and deposition.


Subject(s)
Caenorhabditis elegans Proteins , Caenorhabditis elegans , Subtilisin , Animals , Amino Acid Sequence , Caenorhabditis elegans/metabolism , Caenorhabditis elegans Proteins/genetics , Caenorhabditis elegans Proteins/metabolism , Collagen/genetics , Collagen/metabolism , Proprotein Convertases/genetics , Proprotein Convertases/metabolism , Subtilisin/genetics , Subtilisin/metabolism
3.
Development ; 149(19)2022 10 01.
Article in English | MEDLINE | ID: mdl-36052683

ABSTRACT

The hippocampus is associated with essential brain functions, such as learning and memory. Human hippocampal volume is significantly greater than expected compared with that of non-human apes, suggesting a recent expansion. Intermediate progenitors, which are able to undergo multiple rounds of proliferative division before a final neurogenic division, may have played a role in evolutionary hippocampal expansion. To investigate the evolution of gene regulatory networks underpinning hippocampal neurogenesis in apes, we leveraged the differentiation of human and chimpanzee induced pluripotent stem cells into TBR2 (or EOMES)-positive hippocampal intermediate progenitor cells (hpIPCs). We found that the gene networks active in hpIPCs are significantly different between humans and chimpanzees, with ∼2500 genes being differentially expressed. We demonstrate that species-specific transposon-derived enhancers contribute to these transcriptomic differences. Young transposons, predominantly endogenous retroviruses and SINE-Vntr-Alus (SVAs), were co-opted as enhancers in a species-specific manner. Human-specific SVAs provided substrates for thousands of novel TBR2-binding sites, and CRISPR-mediated repression of these SVAs attenuated the expression of ∼25% of the genes that are upregulated in human intermediate progenitors relative to the same cell population in the chimpanzee.


Subject(s)
DNA Transposable Elements , Pan troglodytes , Animals , DNA Transposable Elements/genetics , Gene Regulatory Networks , Hippocampus , Humans , Neurogenesis , Pan troglodytes/genetics
4.
Development ; 148(19)2021 10 01.
Article in English | MEDLINE | ID: mdl-34423346

ABSTRACT

During convergent differentiation, multiple developmental lineages produce a highly similar or identical cell type. However, few molecular players that drive convergent differentiation are known. Here, we show that the C. elegans Forkhead transcription factor UNC-130 is required in only one of three convergent lineages that produce the same glial cell type. UNC-130 acts transiently as a repressor in progenitors and newly-born terminal cells to allow the proper specification of cells related by lineage rather than by cell type or function. Specification defects correlate with UNC-130:DNA binding, and UNC-130 can be functionally replaced by its human homolog, the neural crest lineage determinant FoxD3. We propose that, in contrast to terminal selectors that activate cell type-specific transcriptional programs in terminally differentiating cells, UNC-130 acts early and specifically in one convergent lineage to produce a cell type that also arises from molecularly distinct progenitors in other lineages.


Subject(s)
Caenorhabditis elegans Proteins/metabolism , Cell Lineage , Neuroglia/metabolism , Transcription Factors/metabolism , Animals , Caenorhabditis elegans , Caenorhabditis elegans Proteins/genetics , Cell Differentiation , Forkhead Transcription Factors/genetics , Forkhead Transcription Factors/metabolism , HEK293 Cells , Humans , Neuroglia/cytology , Transcription Factors/genetics
5.
Cell ; 139(3): 623-33, 2009 Oct 30.
Article in English | MEDLINE | ID: mdl-19879847

ABSTRACT

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.


Subject(s)
Caenorhabditis elegans/cytology , Caenorhabditis elegans/genetics , Cell Lineage , Gene Expression Profiling , Animals , Caenorhabditis elegans/metabolism , Caenorhabditis elegans Proteins , Cell Differentiation , Gene Expression Profiling/methods
6.
Nat Methods ; 15(7): 539-542, 2018 07.
Article in English | MEDLINE | ID: mdl-29941873

ABSTRACT

In single-cell RNA sequencing (scRNA-seq) studies, only a small fraction of the transcripts present in each cell are sequenced. This leads to unreliable quantification of genes with low or moderate expression, which hinders downstream analysis. To address this challenge, we developed SAVER (single-cell analysis via expression recovery), an expression recovery method for unique molecule index (UMI)-based scRNA-seq data that borrows information across genes and cells to provide accurate expression estimates for all genes.


Subject(s)
High-Throughput Nucleotide Sequencing/methods , RNA/genetics , Single-Cell Analysis/methods , Animals , Base Sequence , Cerebral Cortex/cytology , Gene Expression Profiling/methods , Humans , Mice , RNA/chemistry , Sequence Analysis, RNA/methods , Software
7.
Nature ; 512(7515): 400-5, 2014 Aug 28.
Article in English | MEDLINE | ID: mdl-25164749

ABSTRACT

Discovering the structure and dynamics of transcriptional regulatory events in the genome with cellular and temporal resolution is crucial to understanding the regulatory underpinnings of development and disease. We determined the genomic distribution of binding sites for 92 transcription factors and regulatory proteins across multiple stages of Caenorhabditis elegans development by performing 241 ChIP-seq (chromatin immunoprecipitation followed by sequencing) experiments. Integration of regulatory binding and cellular-resolution expression data produced a spatiotemporally resolved metazoan transcription factor binding map. Using this map, we explore developmental regulatory circuits that encode combinatorial logic at the levels of co-binding and co-expression of transcription factors, characterizing the genomic coverage and clustering of regulatory binding, the binding preferences of, and biological processes regulated by, transcription factors, the global transcription factor co-associations and genomic subdomains that suggest shared patterns of regulation, and identifying key transcription factors and transcription factor co-associations for fate specification of individual lineages and cell types.


Subject(s)
Caenorhabditis elegans/growth & development , Caenorhabditis elegans/genetics , Gene Expression Regulation, Developmental/genetics , Genome, Helminth/genetics , Spatio-Temporal Analysis , Transcription Factors/metabolism , Animals , Binding Sites , Caenorhabditis elegans/cytology , Caenorhabditis elegans/embryology , Caenorhabditis elegans Proteins/metabolism , Cell Lineage , Chromatin Immunoprecipitation , Genomics , Larva/cytology , Larva/genetics , Larva/growth & development , Larva/metabolism , Protein Binding
9.
Methods ; 68(3): 431-6, 2014 Aug 01.
Article in English | MEDLINE | ID: mdl-24835576

ABSTRACT

The spatial and temporal control of transgene expression is an important tool in Caenorhabditis elegans biology. We previously described a method for evoking gene expression in arbitrary cells by using a focused pulsed infrared laser to induce a heat shock response (Churgin et al., 2013). Here we describe detailed methods for building and testing a system for performing single-cell heat shock. Steps include setting up the laser and associated components, coupling the laser beam to a microscope, and testing heat shock protocols. All steps can be carried out using readily available off-the-shelf components.


Subject(s)
Caenorhabditis elegans/genetics , Gene Expression Regulation, Developmental/radiation effects , Heat-Shock Response/radiation effects , Single-Cell Analysis/methods , Animals , Animals, Genetically Modified/genetics , Lasers , Promoter Regions, Genetic/radiation effects , Transgenes
10.
Genome Res ; 21(2): 245-54, 2011 Feb.
Article in English | MEDLINE | ID: mdl-21177963

ABSTRACT

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.


Subject(s)
Caenorhabditis elegans/genetics , Caenorhabditis elegans/metabolism , Oligonucleotide Array Sequence Analysis , Transcription Factors/metabolism , Animals , Binding Sites/genetics , Caenorhabditis elegans/cytology , Cluster Analysis , Gene Expression Profiling , Gene Expression Regulation , Models, Theoretical , Molecular Sequence Data , RNA, Untranslated/metabolism , Transcription Factors/genetics , Transcription Initiation Site
11.
Development ; 138(16): 3545-55, 2011 Aug.
Article in English | MEDLINE | ID: mdl-21771815

ABSTRACT

Receptor tyrosine kinases and Notch are crucial for tube formation and branching morphogenesis in many systems, but the specific cellular processes that require signaling are poorly understood. Here we describe sequential roles for Notch and Epidermal growth factor (EGF)-Ras-ERK signaling in the development of epithelial tube cells in the C. elegans excretory (renal-like) organ. This simple organ consists of three tandemly connected unicellular tubes: the excretory canal cell, duct and G1 pore. lin-12 and glp-1/Notch are required to generate the canal cell, which is a source of LIN-3/EGF ligand and physically attaches to the duct during de novo epithelialization and tubulogenesis. Canal cell asymmetry and let-60/Ras signaling influence which of two equivalent precursors will attach to the canal cell. Ras then specifies duct identity, inducing auto-fusion and a permanent epithelial character; the remaining precursor becomes the G1 pore, which eventually loses epithelial character and withdraws from the organ to become a neuroblast. Ras continues to promote subsequent aspects of duct morphogenesis and differentiation, and acts primarily through Raf-ERK and the transcriptional effectors LIN-1/Ets and EOR-1. These results reveal multiple genetically separable roles for Ras signaling in tube development, as well as similarities to Ras-mediated control of branching morphogenesis in more complex organs, including the mammalian kidney. The relative simplicity of the excretory system makes it an attractive model for addressing basic questions about how cells gain or lose epithelial character and organize into tubular networks.


Subject(s)
Caenorhabditis elegans Proteins/metabolism , Caenorhabditis elegans/growth & development , Caenorhabditis elegans/metabolism , Receptors, Notch/metabolism , ras Proteins/metabolism , Animals , Caenorhabditis elegans/cytology , Caenorhabditis elegans Proteins/genetics , Cell Lineage , Gene Expression Regulation, Developmental , MAP Kinase Signaling System , SOS1 Protein/genetics , SOS1 Protein/metabolism , ras Proteins/genetics
12.
Dev Biol ; 366(2): 298-307, 2012 Jun 15.
Article in English | MEDLINE | ID: mdl-22537498

ABSTRACT

Cells perform wide varieties of functions that are facilitated, in part, by adopting unique shapes. Many of the genes and pathways that promote cell fate specification have been elucidated. However, relatively few transcription factors have been identified that promote shape acquisition after fate specification. Here we show that the Nkx5/HMX homeodomain protein MLS-2 is required for cellular elongation and shape maintenance of two tubular epithelial cells in the C. elegans excretory system, the duct and pore cells. The Nkx5/HMX family is highly conserved from sea urchins to humans, with known roles in neuronal and glial development. MLS-2 is expressed in the duct and pore, and defects in mls-2 mutants first arise when the duct and pore normally adopt unique shapes. MLS-2 cooperates with the EGF-Ras-ERK pathway to turn on the LIN-48/Ovo transcription factor in the duct cell during morphogenesis. These results reveal a novel interaction between the Nkx5/HMX family and the EGF-Ras pathway and implicate a transcription factor, MLS-2, as a regulator of cell shape.


Subject(s)
Caenorhabditis elegans Proteins/physiology , Caenorhabditis elegans/physiology , Cell Shape , Epithelial Cells/cytology , Homeodomain Proteins/physiology , Animals , Caenorhabditis elegans/cytology , Caenorhabditis elegans/embryology , Cell Differentiation , Epithelial Cells/physiology , Gene Expression Regulation, Developmental , Morphogenesis , Signal Transduction , Transcription Factors/physiology
13.
PLoS Genet ; 6(9): e1001089, 2010 Sep 02.
Article in English | MEDLINE | ID: mdl-20824072

ABSTRACT

MicroRNAs (miRNAs) have been found to regulate gene expression across eukaryotic species, but the function of most miRNA genes remains unknown. Here we describe how the analysis of the expression patterns of a well-conserved miRNA gene, mir-57, at cellular resolution for every minute during early development of Caenorhabditis elegans provided key insights in understanding its function. Remarkably, mir-57 expression shows strong positional bias but little tissue specificity, a pattern reminiscent of Hox gene function. Despite the minor defects produced by a loss of function mutation, overexpression of mir-57 causes dramatic posterior defects, which also mimic the phenotypes of mutant alleles of a posterior Hox gene, nob-1, an Abd homolog. More importantly, nob-1 expression is found in the same two posterior AB sublineages as those expressing mir-57 but with an earlier onset. Intriguingly, nob-1 functions as an activator for mir-57 expression; it is also a direct target of mir-57. In agreement with this, loss of mir-57 function partially rescues the nob-1 allele defects, indicating a negative feedback regulatory loop between the miRNA and Hox gene to provide positional cues. Given the conservation of the miRNA and Hox gene, the regulatory mechanism might be broadly used across species. The strategy used here to explore mir-57 function provides a path to dissect the regulatory relationship between genes.


Subject(s)
Body Patterning/genetics , Caenorhabditis elegans Proteins/genetics , Caenorhabditis elegans/embryology , Caenorhabditis elegans/genetics , Gene Expression Regulation, Developmental , Homeodomain Proteins/genetics , MicroRNAs/genetics , Transcription Factors/genetics , Animals , Animals, Genetically Modified , Base Sequence , Caenorhabditis elegans/cytology , Caenorhabditis elegans Proteins/metabolism , Cell Lineage , Down-Regulation/genetics , Genes, Helminth/genetics , Genetic Loci/genetics , Homeodomain Proteins/metabolism , MicroRNAs/chemistry , MicroRNAs/metabolism , Molecular Sequence Data , Nucleic Acid Conformation , Organ Specificity/genetics , Phenotype , Promoter Regions, Genetic/genetics , RNA Interference , Time Factors , Transcription Factors/metabolism
14.
PLoS Genet ; 6(2): e1000848, 2010 Feb 19.
Article in English | MEDLINE | ID: mdl-20174564

ABSTRACT

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.


Subject(s)
Caenorhabditis elegans Proteins/metabolism , Caenorhabditis elegans/growth & development , Caenorhabditis elegans/genetics , Environment , Genome, Helminth/genetics , Trans-Activators/metabolism , Animals , Binding Sites , Caenorhabditis elegans Proteins/genetics , Chromatin Immunoprecipitation , Embryo, Nonmammalian/metabolism , Gene Expression Regulation, Developmental , Genes, Helminth/genetics , Green Fluorescent Proteins/metabolism , Larva/metabolism , Protein Binding , RNA Polymerase II/metabolism , Recombinant Fusion Proteins/metabolism , Starvation , Survival Analysis , Trans-Activators/genetics , Transcription Factors/metabolism
15.
bioRxiv ; 2023 Jun 07.
Article in English | MEDLINE | ID: mdl-37333289

ABSTRACT

Some types of collagens, including transmembrane MACIT collagens and C. elegans cuticle collagens, are N-terminally cleaved at a dibasic site that resembles the consensus for furin or other proprotein convertases of the subtilisin/kexin (PCSK) family. Such cleavage may release transmembrane collagens from the plasma membrane and affect extracellular matrix assembly or structure. However, the functional consequences of such cleavage are unclear and evidence for the role of specific PCSKs is lacking. Here, we used endogenous collagen fusions to fluorescent proteins to visualize the secretion and assembly of the first collagen-based cuticle in C. elegans and then tested the role of the PCSK BLI-4 in these processes. Unexpectedly, we found that cuticle collagens SQT-3 and DPY-17 are secreted into the extraembryonic space several hours before cuticle matrix assembly. Furthermore, this early secretion depends on BLI-4/PCSK; in bli-4 and cleavage-site mutants, SQT-3 and DPY-17 are not efficiently secreted and instead form large intracellular aggregates. Their later assembly into cuticle matrix is reduced but not entirely blocked. These data reveal a role for collagen N-terminal processing in intracellular trafficking and in the spatial and temporal restriction of matrix assembly in vivo . Our observations also prompt a revision of the classic model for C. elegans cuticle matrix assembly and the pre-cuticle-to-cuticle transition, suggesting that cuticle layer assembly proceeds via a series of regulated steps and not simply by sequential secretion and deposition.

16.
Ann N Y Acad Sci ; 1506(1): 74-97, 2021 12.
Article in English | MEDLINE | ID: mdl-34605044

ABSTRACT

Single cell biology has the potential to elucidate many critical biological processes and diseases, from development and regeneration to cancer. Single cell analyses are uncovering the molecular diversity of cells, revealing a clearer picture of the variation among and between different cell types. New techniques are beginning to unravel how differences in cell state-transcriptional, epigenetic, and other characteristics-can lead to different cell fates among genetically identical cells, which underlies complex processes such as embryonic development, drug resistance, response to injury, and cellular reprogramming. Single cell technologies also pose significant challenges relating to processing and analyzing vast amounts of data collected. To realize the potential of single cell technologies, new computational approaches are needed. On March 17-19, 2021, experts in single cell biology met virtually for the Keystone eSymposium "Single Cell Biology" to discuss advances both in single cell applications and technologies.


Subject(s)
Cell Differentiation/physiology , Cellular Reprogramming/physiology , Congresses as Topic/trends , Embryonic Development/physiology , Research Report , Single-Cell Analysis/trends , Animals , Cell Lineage/physiology , Humans , Macrophages/physiology , Single-Cell Analysis/methods
17.
BMC Bioinformatics ; 11: 84, 2010 Feb 11.
Article in English | MEDLINE | ID: mdl-20146825

ABSTRACT

BACKGROUND: Image analysis is an essential component in many biological experiments that study gene expression, cell cycle progression, and protein localization. A protocol for tracking the expression of individual C. elegans genes was developed that collects image samples of a developing embryo by 3-D time lapse microscopy. In this protocol, a program called StarryNite performs the automatic recognition of fluorescently labeled cells and traces their lineage. However, due to the amount of noise present in the data and due to the challenges introduced by increasing number of cells in later stages of development, this program is not error free. In the current version, the error correction (i.e., editing) is performed manually using a graphical interface tool named AceTree, which is specifically developed for this task. For a single experiment, this manual annotation task takes several hours. RESULTS: In this paper, we reduce the time required to correct errors made by StarryNite. We target one of the most frequent error types (movements annotated as divisions) and train a support vector machine (SVM) classifier to decide whether a division call made by StarryNite is correct or not. We show, via cross-validation experiments on several benchmark data sets, that the SVM successfully identifies this type of error significantly. A new version of StarryNite that includes the trained SVM classifier is available at http://starrynite.sourceforge.net. CONCLUSIONS: We demonstrate the utility of a machine learning approach to error annotation for StarryNite. In the process, we also provide some general methodologies for developing and validating a classifier with respect to a given pattern recognition task.


Subject(s)
Artificial Intelligence , Caenorhabditis elegans/embryology , Caenorhabditis elegans/genetics , Embryo, Nonmammalian/metabolism , Imaging, Three-Dimensional/methods , Animals , Caenorhabditis elegans Proteins/genetics , Caenorhabditis elegans Proteins/metabolism , Gene Expression Profiling/methods , Image Interpretation, Computer-Assisted/methods
18.
G3 (Bethesda) ; 10(6): 1949-1962, 2020 06 01.
Article in English | MEDLINE | ID: mdl-32273286

ABSTRACT

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.


Subject(s)
Caenorhabditis elegans Proteins , Caenorhabditis elegans , Animals , Basic Helix-Loop-Helix Transcription Factors/genetics , Basic Helix-Loop-Helix Transcription Factors/metabolism , Caenorhabditis elegans/genetics , Caenorhabditis elegans/metabolism , Caenorhabditis elegans Proteins/genetics , Caenorhabditis elegans Proteins/metabolism , Gene Expression Regulation, Developmental , Nervous System/metabolism , Neurons/metabolism , Transcription Factors/genetics
19.
Dev Biol ; 318(1): 65-72, 2008 Jun 01.
Article in English | MEDLINE | ID: mdl-18430415

ABSTRACT

As a fundamental process of development, cell proliferation must be coordinated with other processes such as fate differentiation. Through statistical analysis of individual cell cycle lengths of the first 8 out of 10 rounds of embryonic cell division in Caenorhabditis elegans, we identified synchronous and invariantly ordered divisions that are tightly associated with fate differentiation. Our results suggest a three-tier model for fate control of cell cycle pace: the primary control of cell cycle pace is established by lineage and the founder cell fate, then fine-tuned by tissue and organ differentiation within each lineage, then further modified by individualization of cells as they acquire unique morphological and physiological roles in the variant body plan. We then set out to identify the pace-setting mechanisms in different fates. Our results suggest that ubiquitin-mediated degradation of CDC-25.1 is a rate-determining step for the E (gut) and P(3) (muscle and germline) lineages but not others, even though CDC-25.1 and its apparent decay have been detected in all lineages. Our results demonstrate the power of C. elegans embryogenesis as a model to dissect the interaction between differentiation and proliferation, and an effective approach combining genetic and statistical analysis at single-cell resolution.


Subject(s)
Caenorhabditis elegans Proteins/metabolism , Caenorhabditis elegans/embryology , Caenorhabditis elegans/physiology , Cell Cycle/physiology , Cell Differentiation/physiology , Embryonic Development/physiology , Animals , Caenorhabditis elegans/anatomy & histology , Caenorhabditis elegans Proteins/genetics , Cell Lineage , Female , cdc25 Phosphatases/genetics , cdc25 Phosphatases/metabolism
20.
Dev Biol ; 314(1): 93-9, 2008 Feb 01.
Article in English | MEDLINE | ID: mdl-18164284

ABSTRACT

Comparative genomic analysis of important signaling pathways in Caenorhabditis briggsae and Caenorhabditis elegans reveals both conserved features and also differences. To build a framework to address the significance of these features we determined the C. briggsae embryonic cell lineage, using the tools StarryNite and AceTree. We traced both cell divisions and cell positions for all cells through all but the last round of cell division and for selected cells through the final round. We found the lineage to be remarkably similar to that of C. elegans. Not only did the founder cells give rise to similar numbers of progeny, the relative cell division timing and positions were largely maintained. These lineage similarities appear to give rise to similar cell fates as judged both by the positions of lineally equivalent cells and by the patterns of cell deaths in both species. However, some reproducible differences were seen, e.g., the P4 cell cycle length is more than 40% longer in C. briggsae than that in C. elegans (p<0.01). The extensive conservation of embryonic development between such divergent species suggests that substantial evolutionary distance between these two species has not altered these early developmental cellular events, although the developmental defects of transpecies hybrids suggest that the details of the underlying molecular pathways have diverged sufficiently so as to not be interchangeable.


Subject(s)
Caenorhabditis/embryology , Cell Lineage/physiology , Animals , Biological Evolution , Caenorhabditis/cytology , Caenorhabditis elegans/cytology , Caenorhabditis elegans/embryology , Cell Death/physiology , Cell Movement/physiology , Embryo, Nonmammalian/cytology , Phylogeny , Signal Transduction , Species Specificity
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