computational-analysis-caenorhabditis-elegans-germline-to-study

Computational Analysis of the <em>Caenorhabditis elegans</em> Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...

Monash University

The ability of animals to sense and respond to elevated temperature is essential for survival. Transcriptional control of the heat stress response has been much studied, whereas its posttranscriptional regulation by microRNAs (miRNAs) is not well understood. Here we analyzed the miRNA response to heat stress in Caenorhabditis elegans and show that a discrete subset of miRNAs is thermoregulated. Using in-depth phenotypic analyses of miRNA deletion mutant strains we reveal multiple developmental and post-developmental survival and behavioral functions for specific miRNAs during heat stress. We have identified additional functions for already known players (mir-71 and mir-239) as well as identifying mir-80 and the mir-229 mir-64-66 cluster as important regulators of the heat stress response in C. elegans. These findings uncover an additional layer of complexity to the regulation of stress signaling that enables animals to robustly respond to the changing environment. 

Specific microRNAs regulate heat stress responses in Caenorhabditis elegans.

Layered strategies to combat hypoxia provide flexibility in dynamic oxygen environments. Here we show that multiple miRNAs are required for hypoxic survival responses during C. elegans embryogenesis. Certain miRNAs promote while others antagonize the hypoxic survival response. We found that expression of the mir-35 family is regulated by hypoxia in a HIF-1-independent manner and loss of mir-35-41 weakens hypoxic survival mechanisms in embryos. In addition, correct regulation of the RNA binding protein, SUP-26, a mir-35 family target, is needed for survival in chronic hypoxia. The identification of the full mRNA target repertoire of these miRNAs will reveal the miRNA-regulated network of hypoxic survival mechanisms in C. elegans. 

microRNA regulation of the embryonic hypoxic response in Caenorhabditis elegans.

MicroRNAs (miRNAs) are a class of short non-coding RNAs that operate as prominent post-transcriptional regulators of eukaryotic gene expression. miRNAs are abundantly expressed in the brain of most animals and exert diverse roles. The anatomical and functional complexity of the brain requires the precise coordination of multilayered gene regulatory networks. The flexibility, speed, and reversibility of miRNA function provide precise temporal and spatial gene regulatory capabilities that are crucial for the correct functioning of the brain. Studies have shown that the underlying molecular mechanisms controlled by miRNAs in the nervous systems of invertebrate and vertebrate models are remarkably conserved in humans. We endeavor to provide insight into the roles of miRNAs in the nervous systems of these model organisms and discuss how such information may be used to inform regarding diseases of the human brain. 

MicroRNAs: Not "Fine-Tuners" but Key Regulators of Neuronal Development and Function.

Cytosolic calcium is an important factor during fertilization, development and differentiation. Hence, the control of cytosolic calcium levels has been studied extensively for several decades. Numerous calcium channels have been identified and their mechanism of action elucidated. However, the mode of calcium channel regulation remains elusive. Here we discuss our recent findings regarding the role of syndecans in the regulation of cytosolic calcium levels. Syndecans are transmembrane proteoglycans present in both vertebrates and invertebrates that interact with extracellular ligands resulting in the activation of several downstream signaling pathways. We identified a previously unappreciated role of syndecans in cytosolic calcium regulation in mammals that is conserved in C. elegans. We concluded that calcium regulation is the basic, evolutionarily conserved role for syndecans, which enables them to be integral for multiple cellular functions. 

Redefining the role of syndecans in C. elegans biology.

Cell-extracellular matrix (ECM) and cell-cell junctions that employ microfilaments are sites of tension. They are important for tissue repair, morphogenetic movements and can be emblematic of matrix contraction in fibrotic disease and the stroma of solid tumors. One cell surface receptor, syndecan-4, has been shown to regulate focal adhesions, junctions that form at the ends of microfilament bundles in response to matrix components such as fibronectin. Recently it has been shown that signaling emanating from this proteoglycan receptor includes regulation of Rho family GTPases and cytosolic calcium. While it is known that cell-ECM and cell-cell junctions may be linked, possible roles for syndecans in this process are not understood. Here we show that wild type primary fibroblasts and those lacking syndecan-4 utilize different cadherins in their adherens junctions and that tension is a major factor in this differential response. This corresponds to the reduced ability of fibroblasts lacking syndecan-4 to exert tension on the ECM and we now show that this may extend to reduced tension in cell-cell adhesion.

Cell-extracellular matrix and cell-cell adhesion are linked by syndecan-4.

Nogo-A is a membrane-bound protein that functions to inhibit neuronal migration, adhesion, and neurite outgrowth during development. In the mature nervous system, Nogo-A stabilizes neuronal wiring to inhibit neuronal plasticity and regeneration after injury. Here, we show that RET-1, the sole Nogo-A homolog in Caenorhabditis elegans, is required to control developmental wiring of a specific subset of neurons. In ret-1 deletion mutant animals, specific ventral nerve cord axons are misguided where they fail to respect the ventral midline boundary. We found that ret-1 is expressed in multiple neurons during development, and, through mosaic analysis, showed that ret-1 controls axon guidance in a cell-autonomous manner. Finally, as in mammals, ret-1 regulates ephrin expression, and dysregulation of the ephrin ligand VAB-2 is partially responsible for the ret-1 mutant axonal defects. Together, our data present a previously unidentified function for RET-1 in the nervous system of C. elegans.

Developmental Wiring of Specific Neurons Is Regulated by RET-1/Nogo-A in Caenorhabditis elegans.

Monitoring of neuronal activity within circuits facilitates integrated responses and rapid changes in behavior. We have identified a system in Caenorhabditis elegans where neuropeptide expression is dependent on the ability of the BAG neurons to sense carbon dioxide. In C. elegans, CO sensing is predominantly coordinated by the BAG-expressed receptor-type guanylate cyclase GCY-9. GCY-9 binding to CO causes accumulation of cyclic GMP and opening of the cGMP-gated TAX-2/TAX-4 cation channels; provoking an integrated downstream cascade that enables C. elegans to avoid high CO. Here we show that cGMP regulation by GCY-9 and the PDE-1 phosphodiesterase controls BAG expression of a FMRFamide-related neuropeptide FLP-19 reporter (flp-19::GFP). This regulation is specific for CO-sensing function of the BAG neurons, as loss of oxygen sensing function does not affect flp-19::GFP expression. We also found that expression of flp-19::GFP is controlled in parallel to GCY-9 by the activity-dependent transcription factor CREB (CRH-1) and the cAMP-dependent protein kinase (KIN-2) signaling pathway. We therefore show that two parallel pathways regulate neuropeptide gene expression in the BAG sensory neurons: the ability to sense changes in carbon dioxide and CREB transcription factor. Such regulation may be required in particular environmental conditions to enable sophisticated behavioral decisions to be performed.

Control of Neuropeptide Expression by Parallel Activity-dependent Pathways in Caenorhabditis elegans.

Animal behavior is shaped through interplay among genes, the environment, and previous experience. As in mammals, satiety signals induce quiescence in  Here we report that the  transcription factor ETS-5, an ortholog of mammalian FEV/Pet1, controls satiety-induced quiescence. Nutritional status has a major influence on  behavior. When foraging, food availability controls behavioral state switching between active (roaming) and sedentary (dwelling) states; however, when provided with high-quality food,  become sated and enter quiescence. We show that ETS-5 acts to promote roaming and inhibit quiescence by setting the internal "satiety quotient" through fat regulation. Acting from the ASG and BAG sensory neurons, we show that ETS-5 functions in a complex network with serotonergic and neuropeptide signaling pathways to control food-regulated behavioral state switching. Taken together, our results identify a neuronal mechanism for controlling intestinal fat stores and organismal behavioral states in , and establish a paradigm for the elucidation of obesity-relevant mechanisms.

The ETS-5 transcription factor regulates activity states in  by controlling satiety.

Development of complex nervous systems requires precisely controlled neurogenesis. The generation and specification of neurons occur through the transcriptional and post-transcriptional control of complex regulatory networks. In vertebrates and invertebrates, the proneural basic-helix-loop-helix (bHLH) family of transcription factors has multiple functions in neurogenesis. Here, we identified the LIN-32/Atonal bHLH transcription factor as a key regulator of URXL/R oxygen-sensing neuron development in Caenorhabditis elegans. When LIN-32/Atonal expression is lost, the expression of URX specification and terminal differentiation genes is abrogated. As such, lin-32 mutant animals are unable to respond to increases in environmental oxygen. The URX neurons are generated from a branch of the cell lineage that also produces the CEPDL/R and URADL/R neurons. We found development of these neurons is also defective, suggesting that LIN-32/Atonal regulates neuronal development of the entire lineage. Finally, our results show that aspects of URX neuronal fate are partially restored in lin-32 mutant animals when the apoptosis pathway is inhibited. This suggests that, as in other organisms, LIN-32/Atonal regulates neuronal apoptosis.

LIN-32/Atonal Controls Oxygen Sensing Neuron Development in Caenorhabditis elegans.

The Caenorhabditis elegans germline is widely used as a model to study stem cell development, chromosome dynamics and apoptosis. Major readouts of germline phenotypes such as cell counting and protein expression profiling are routinely analyzed manually and in a two-dimensional manner. The major disadvantages of the existing approaches are 1) they are time-consuming and laborious and 2) there is an inability to study the effects of genetic mutations in three dimensions. Here, we demonstrate a rapid, automated method for analyzing the three-dimensional distribution of proteins, germline nuclei and cytoskeletal structures in the C. elegans germline. Using this method, we have revealed previously unappreciated germline organization and cytoskeletal structures that will have a major impact on the characterization of germline phenotypes. To conclude, our new method dramatically enhances the efficiency and resolution of C. elegans germline analysis and may be applied to other cellular structures.

Automated three-dimensional reconstruction of the Caenorhabditis elegans germline.

The nematode Caenorhabditis elegans is widely used as a model organism to study cell and developmental biology. Quantitative proteomics of C. elegans is still in its infancy and, so far, most studies have been performed on adult worm samples. Here, we used quantitative mass spectrometry to characterize protein level changes across the four larval developmental stages (L1-L4) of C. elegans. In total, we identified 4130 proteins, and quantified 1541 proteins that were present across all four stages in three biological replicates from independent experiments. Using hierarchical clustering and functional ontological analyses, we identified 21 clusters containing proteins with similar protein profiles across the four stages, and highlighted the most overrepresented biological functions in each of these protein clusters. In addition, we used the dataset to identify putative larval stage-specific proteins in each individual developmental stage, as well as in the early and late developmental stages. In summary, this dataset provides system-wide analysis of protein level changes across the four C. elegans larval developmental stages, which serves as a useful resource for the C. elegans research community. MS data were deposited in ProteomeXchange (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository with the primary accession identifier PXD006676.

Proteomic Characterization of Caenorhabditis elegans Larval Development.

Animals use behavioral strategies to seek optimal environments. Population behavioral assays provide a robust means to determine the effect of genetic perturbations on the ability of animals to sense and respond to changes in the environment. Here, we describe a  population behavioral assay used to measure locomotory responses to changes in environmental oxygen (O) and carbon dioxide (CO) concentrations. These behavioral assays are high-throughput and enable examination of genetic, neuronal and circuit function.

Behavioral Assays to Study Oxygen and Carbon Dioxide Sensing in .

Studies of nervous system connectivity, in a wide variety of species and at different scales of resolution, have identified several highly conserved motifs of network organization. One such motif is a heterogeneous distribution of connectivity across neural elements, such that some elements act as highly connected and functionally important network hubs. These brain network hubs are also densely interconnected, forming a so-called rich club. Recent work in mouse has identified a distinctive transcriptional signature of neural hubs, characterized by tightly coupled expression of oxidative metabolism genes, with similar genes characterizing macroscale inter-modular hub regions of the human cortex. Here, we sought to determine whether hubs of the neuronal C. elegans connectome also show tightly coupled gene expression. Using open data on the chemical and electrical connectivity of 279 C. elegans neurons, and binary gene expression data for each neuron across 948 genes, we computed a correlated gene expression score for each pair of neurons, providing a measure of their gene expression similarity. We demonstrate that connections between hub neurons are the most similar in their gene expression while connections between nonhubs are the least similar. Genes with the greatest contribution to this effect are involved in glutamatergic and cholinergic signaling, and other communication processes. We further show that coupled expression between hub neurons cannot be explained by their neuronal subtype (i.e., sensory, motor, or interneuron), separation distance, chemically secreted neurotransmitter, birth time, pairwise lineage distance, or their topological module affiliation. Instead, this coupling is intrinsically linked to the identity of most hubs as command interneurons, a specific class of interneurons that regulates locomotion. Our results suggest that neural hubs may possess a distinctive transcriptional signature, preserved across scales and species, that is related to the involvement of hubs in regulating the higher-order behaviors of a given organism.

Article	Year
Specific microRNAs regulate heat stress responses in Caenorhabditis elegans. Scientific reports\| PubMed ID: 25746291	2015
microRNA regulation of the embryonic hypoxic response in Caenorhabditis elegans. Scientific reports\| PubMed ID: 26063315	2015
MicroRNAs: Not "Fine-Tuners" but Key Regulators of Neuronal Development and Function. Frontiers in neurology\| PubMed ID: 26635721	2015
Redefining the role of syndecans in C. elegans biology. Worm\| PubMed ID: 27073736	2016
Cell-extracellular matrix and cell-cell adhesion are linked by syndecan-4. Matrix biology : journal of the International Society for Matrix Biology\| PubMed ID: 27751945	2017
Developmental Wiring of Specific Neurons Is Regulated by RET-1/Nogo-A in Caenorhabditis elegans. Genetics\| PubMed ID: 27821431	2017
Control of Neuropeptide Expression by Parallel Activity-dependent Pathways in Caenorhabditis elegans. Scientific reports\| PubMed ID: 28139692	2017
The ETS-5 transcription factor regulates activity states in by controlling satiety. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 28193866	2017
LIN-32/Atonal Controls Oxygen Sensing Neuron Development in Caenorhabditis elegans. Scientific reports\| PubMed ID: 28779171	2017
Automated three-dimensional reconstruction of the Caenorhabditis elegans germline. Developmental biology\| PubMed ID: 29079422	2017
Proteomic Characterization of Caenorhabditis elegans Larval Development. Proteomics\| PubMed ID: 29178193	2018
Behavioral Assays to Study Oxygen and Carbon Dioxide Sensing in . Bio-protocol\| PubMed ID: 29335676	2018
Hub connectivity, neuronal diversity, and gene expression in the Caenorhabditis elegans connectome. PLoS computational biology\| PubMed ID: 29432412	2018

Roger Pocock

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