Predicting Phenotypic Diversity and the Underlying Quantitative Molecular Transitions

During development, signaling networks control the formation of multicellular patterns. To what extent quantitative fluctuations in these complex networks may affect multicellular phenotype remains unclear. Here, we describe a computational approach to predict and analyze the phenotypic diversity that is accessible to a developmental signaling network. Applying this framework to vulval development in C. elegans, we demonstrate that quantitative changes in the regulatory network can render ~500 multicellular phenotypes. This phenotypic capacity is an order-of-magnitude below the theoretical upper limit for this system but yet is large enough to demonstrate that the system is not restricted to a select few outcomes. Using metrics to gauge the robustness of these phenotypes to parameter perturbations, we identify a select subset of novel phenotypes that are the most promising for experimental validation. In addition, our model calculations provide a layout of these phenotypes in network parameter space. Analyzing this landscape of multicellular phenotypes yielded two significant insights. First, we show that experimentally well-established mutant phenotypes may be rendered using non-canonical network perturbations. Second, we show that the predicted multicellular patterns include not only those observed in C. elegans, but also those occurring exclusively in other species of the Caenorhabditis genus. This result demonstrates that quantitative diversification of a common regulatory network is indeed demonstrably sufficient to generate the phenotypic differences observed across three major species within the Caenorhabditis genus. Using our computational framework, we systematically identify the quantitative changes that may have occurred in the regulatory network during the evolution of these species. Our model predictions show that significant phenotypic diversity may be sampled through quantitative variations in the regulatory network without overhauling the core network architecture. Furthermore, by comparing the predicted landscape of phenotypes to multicellular patterns that have been experimentally observed across multiple species, we systematically trace the quantitative regulatory changes that may have occurred during the evolution of the Caenorhabditis genus

The Roles and Acting Mechanism of Caenorhabditis elegans DNase II Genes in Apoptotic DNA Degradation and Development

DNase II enzymes are acidic endonucleases that have been implicated in mediating apoptotic DNA degradation, a critical cell death execution event. C. elegans genome contains three DNase II homologues, NUC-1, CRN-6, and CRN-7, but their expression patterns, acting sites, and roles in apoptotic DNA degradation and development are unclear. We have conducted a comprehensive analysis of three C. elegans DNase II genes and found that nuc-1 plays a major role, crn-6 plays an auxiliary role, and crn-7 plays a negligible role in resolving 3′ OH DNA breaks generated in apoptotic cells. Promoter swapping experiments suggest that crn-6 but not crn-7 can partially substitute for nuc-1 in mediating apoptotic DNA degradation and both fail to replace nuc-1 in degrading bacterial DNA in intestine. Despite of their restricted and largely non-overlapping expression patterns, both CRN-6 and NUC-1 can mediate apoptotic DNA degradation in many cells, suggesting that they are likely secreted nucleases that are retaken up by other cells to exert DNA degradation functions. Removal or disruption of NUC-1 secretion signal eliminates NUC-1's ability to mediate DNA degradation across its expression border. Furthermore, blocking cell corpse engulfment does not affect apoptotic DNA degradation mediated by nuc-1, suggesting that NUC-1 acts in apoptotic cells rather than in phagocytes to resolve 3′ OH DNA breaks. Our study illustrates how multiple DNase II nucleases play differential roles in apoptotic DNA degradation and development and reveals an unexpected mode of DNase II action in mediating DNA degradation

A Genome-Wide RNAi Screen for Factors Involved in Neuronal Specification in Caenorhabditis elegans

One of the central goals of developmental neurobiology is to describe and understand the multi-tiered molecular events that control the progression of a fertilized egg to a terminally differentiated neuron. In the nematode Caenorhabditis elegans, the progression from egg to terminally differentiated neuron has been visually traced by lineage analysis. For example, the two gustatory neurons ASEL and ASER, a bilaterally symmetric neuron pair that is functionally lateralized, are generated from a fertilized egg through an invariant sequence of 11 cellular cleavages that occur stereotypically along specific cleavage planes. Molecular events that occur along this developmental pathway are only superficially understood. We take here an unbiased, genome-wide approach to identify genes that may act at any stage to ensure the correct differentiation of ASEL. Screening a genome-wide RNAi library that knocks-down 18,179 genes (94% of the genome), we identified 245 genes that affect the development of the ASEL neuron, such that the neuron is either not generated, its fate is converted to that of another cell, or cells from other lineage branches now adopt ASEL fate. We analyze in detail two factors that we identify from this screen: (1) the proneural gene hlh-14, which we find to be bilaterally expressed in the ASEL/R lineages despite their asymmetric lineage origins and which we find is required to generate neurons from several lineage branches including the ASE neurons, and (2) the COMPASS histone methyltransferase complex, which we find to be a critical embryonic inducer of ASEL/R asymmetry, acting upstream of the previously identified miRNA lsy-6. Our study represents the first comprehensive, genome-wide analysis of a single neuronal cell fate decision. The results of this analysis provide a starting point for future studies that will eventually lead to a more complete understanding of how individual neuronal cell types are generated from a single-cell embryo

Bone marrow adipose tissue is a unique adipose subtype with distinct roles in glucose homeostasis

Bone marrow adipose tissue (BMAT) comprises >10% of total adipose mass, yet unlike white or brown adipose tissues (WAT or BAT) its metabolic functions remain unclear. Herein, we address this critical gap in knowledge. Our transcriptomic analyses revealed that BMAT is distinct from WAT and BAT, with altered glucose metabolism and decreased insulin responsiveness. We therefore tested these functions in mice and humans using positron emission tomography-computed tomography (PET/CT) with 18F-fluorodeoxyglucose. This revealed that BMAT resists insulin- and cold-stimulated glucose uptake, while further in vivo studies showed that, compared to WAT, BMAT resists insulin-stimulated Akt phosphorylation. Thus, BMAT is functionally distinct from WAT and BAT. However, in humans basal glucose uptake in BMAT is greater than in axial bones or subcutaneous WAT and can be greater than that in skeletal muscle, underscoring the potential of BMAT to influence systemic glucose homeostasis. These PET/CT studies characterise BMAT function in vivo, establish new methods for BMAT analysis, and identify BMAT as a distinct, major adipose tissue subtype

Caenorhabditis elegans Cyclin D/CDK4 and Cyclin E/CDK2 Induce Distinct Cell Cycle Re-Entry Programs in Differentiated Muscle Cells

Cell proliferation and differentiation are regulated in a highly coordinated and inverse manner during development and tissue homeostasis. Terminal differentiation usually coincides with cell cycle exit and is thought to engage stable transcriptional repression of cell cycle genes. Here, we examine the robustness of the post-mitotic state, using Caenorhabditis elegans muscle cells as a model. We found that expression of a G1 Cyclin and CDK initiates cell cycle re-entry in muscle cells without interfering with the differentiated state. Cyclin D/CDK4 (CYD-1/CDK-4) expression was sufficient to induce DNA synthesis in muscle cells, in contrast to Cyclin E/CDK2 (CYE-1/CDK-2), which triggered mitotic events. Tissue-specific gene-expression profiling and single molecule FISH experiments revealed that Cyclin D and E kinases activate an extensive and overlapping set of cell cycle genes in muscle, yet failed to induce some key activators of G1/S progression. Surprisingly, CYD-1/CDK-4 also induced an additional set of genes primarily associated with growth and metabolism, which were not activated by CYE-1/CDK-2. Moreover, CYD-1/CDK-4 expression also down-regulated a large number of genes enriched for catabolic functions. These results highlight distinct functions for the two G1 Cyclin/CDK complexes and reveal a previously unknown activity of Cyclin D/CDK-4 in regulating metabolic gene expression. Furthermore, our data demonstrate that many cell cycle genes can still be transcriptionally induced in post-mitotic muscle cells, while maintenance of the post-mitotic state might depend on stable repression of a limited number of critical cell cycle regulators

Caenorhabditis elegans Battling Starvation Stress: Low Levels of Ethanol Prolong Lifespan in L1 Larvae

The nematode Caenorhabditis elegans arrests development at the first larval stage if food is not present upon hatching. Larvae in this stage provide an excellent model for studying stress responses during development. We found that supplementing starved larvae with ethanol markedly extends their lifespan within this L1 diapause. The effects of ethanol-induced lifespan extension can be observed when the ethanol is added to the medium at any time between 0 and 10 days after hatching. The lowest ethanol concentration that extended lifespan was 1 mM (0.005%); higher concentrations to 68 mM (0.4%) did not result in increased survival. In spite of their extended survival, larvae did not progress to the L2 stage. Supplementing starved cultures with n-propanol and n-butanol also extended lifespan, but methanol and isopropanol had no measurable effect. Mass spectrometry analysis of nematode fatty acids and amino acids revealed that L1 larvae can incorporate atoms from ethanol into both types of molecules. Based on these data, we suggest that ethanol supplementation may extend the lifespan of L1 larvae by either serving as a carbon and energy source and/or by inducing a stress response

A Negative Regulatory Loop between MicroRNA and Hox Gene Controls Posterior Identities in Caenorhabditis elegans

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

Visualization of C. elegans transgenic arrays by GFP

BACKGROUND: Targeting the green fluorescent protein (GFP) via the E. coli lac repressor (LacI) to a specific DNA sequence, the lac operator (lacO), allows visualization of chromosomes in yeast and mammalian cells. In principle this method of visualization could be used for genetic mosaic analysis, which requires cell-autonomous markers that can be scored easily and at single cell resolution. The C. elegans lin-3 gene encodes an epidermal growth factor family (EGF) growth factor. lin-3 is expressed in the gonadal anchor cell and acts through LET-23 (transmembrane protein tyrosine kinase and ortholog of EGF receptor) to signal the vulval precursor cells to generate vulval tissue. lin-3 is expressed in the vulval cells later, and recent evidence raises the possibility that lin-3 acts in the vulval cells as a relay signal during vulval induction. It is thus of interest to test the site of action of lin-3 by mosaic analysis. RESULTS: We visualized transgenes in living C. elegans by targeting the green fluorescent protein (GFP) via the E. coli lac repressor (LacI) to a specific 256 sequence repeat of the lac operator (lacO) incorporated into transgenes. We engineered animals to express a nuclear-localized GFP-LacI fusion protein. C. elegans cells having a lacO transgene result in nuclear-localized bright spots (i.e., GFP-LacI bound to lacO). Cells with diffuse nuclear fluorescence correspond to unbound nuclear localized GFP-LacI. We detected chromosomes in living animals by chromosomally integrating the array of the lacO repeat sequence and visualizing the integrated transgene with GFP-LacI. This detection system can be applied to determine polyploidy as well as investigating chromosome segregation. To assess the GFP-LacI•lacO system as a marker for mosaic analysis, we conducted genetic mosaic analysis of the epidermal growth factor lin-3, expressed in the anchor cell. We establish that lin-3 acts in the anchor cell to induce vulva development, demonstrating this method's utility in detecting the presence of a transgene. CONCLUSION: The GFP-LacI•lacO transgene detection system works in C. elegans for visualization of chromosomes and extrachromosomal transgenes. It can be used as a marker for genetic mosaic analysis. The lacO repeat sequence as an extrachromosomal array becomes a valuable technique allowing rapid, accurate determination of spontaneous loss of the array, thereby allowing high-resolution mosaic analysis. The lin-3 gene is required in the anchor cell to induce the epidermal vulval precursors cells to undergo vulval development

An Integrated Strategy to Study Muscle Development and Myofilament Structure in Caenorhabditis elegans

A crucial step in the development of muscle cells in all metazoan animals is the assembly and anchorage of the sarcomere, the essential repeat unit responsible for muscle contraction. In Caenorhabditis elegans, many of the critical proteins involved in this process have been uncovered through mutational screens focusing on uncoordinated movement and embryonic arrest phenotypes. We propose that additional sarcomeric proteins exist for which there is a less severe, or entirely different, mutant phenotype produced in their absence. We have used Serial Analysis of Gene Expression (SAGE) to generate a comprehensive profile of late embryonic muscle gene expression. We generated two replicate long SAGE libraries for sorted embryonic muscle cells, identifying 7,974 protein-coding genes. A refined list of 3,577 genes expressed in muscle cells was compiled from the overlap between our SAGE data and available microarray data. Using the genes in our refined list, we have performed two separate RNA interference (RNAi) screens to identify novel genes that play a role in sarcomere assembly and/or maintenance in either embryonic or adult muscle. To identify muscle defects in embryos, we screened specifically for the Pat embryonic arrest phenotype. To visualize muscle defects in adult animals, we fed dsRNA to worms producing a GFP-tagged myosin protein, thus allowing us to analyze their myofilament organization under gene knockdown conditions using fluorescence microscopy. By eliminating or severely reducing the expression of 3,300 genes using RNAi, we identified 122 genes necessary for proper myofilament organization, 108 of which are genes without a previously characterized role in muscle. Many of the genes affecting sarcomere integrity have human homologs for which little or nothing is known

C. elegans Germ Cells Show Temperature and Age-Dependent Expression of Cer1, a Gypsy/Ty3-Related Retrotransposon

Virus-like particles (VLPs) have not been observed in Caenorhabditis germ cells, although nematode genomes contain low numbers of retrotransposon and retroviral sequences. We used electron microscopy to search for VLPs in various wild strains of Caenorhabditis, and observed very rare candidate VLPs in some strains, including the standard laboratory strain of C. elegans, N2. We identified the N2 VLPs as capsids produced by Cer1, a retrotransposon in the Gypsy/Ty3 family of retroviruses/retrotransposons. Cer1 expression is age and temperature dependent, with abundant expression at 15°C and no detectable expression at 25°C, explaining how VLPs escaped detection in previous studies. Similar age and temperature-dependent expression of Cer1 retrotransposons was observed for several other wild strains, indicating that these properties are common, if not integral, features of this retroelement. Retrotransposons, in contrast to DNA transposons, have a cytoplasmic stage in replication, and those that infect non-dividing cells must pass their genomic material through nuclear pores. In most C. elegans germ cells, nuclear pores are largely covered by germline-specific organelles called P granules. Our results suggest that Cer1 capsids target meiotic germ cells exiting pachytene, when free nuclear pores are added to the nuclear envelope and existing P granules begin to be removed. In pachytene germ cells, Cer1 capsids concentrate away from nuclei on a subset of microtubules that are exceptionally resistant to microtubule inhibitors; the capsids can aggregate these stable microtubules in older adults, which exhibit a temperature-dependent decrease in egg viability. When germ cells exit pachytene, the stable microtubules disappear and capsids redistribute close to nuclei that have P granule-free nuclear pores. This redistribution is microtubule dependent, suggesting that capsids that are released from stable microtubules transfer onto new, dynamic microtubules to track toward nuclei. These studies introduce C. elegans as a model to study the interplay between retroelements and germ cell biology