Wendy Hanna-Rose (2001) International C. elegans Meeting "Organogenesis of the C. elegans vulva: cog mutants"

Wendy Hanna-Rose

[International C. elegans Meeting, 2001]

While a large body of research has provided insight into how cell fate is specified early in embryogenesis, little is known about mechanisms through which these cell fate decisions are implemented, ultimately resulting in the formation of discrete organs. C. elegans vulval development provides a powerful model system for elucidating the organogenesis process. Our research focuses on the aspect of how the developing vulva connects properly to the overlying uterus. We have been continuing the studies of cog-2 and egl-26 , which display Cog ( co nnection of g onad defective) phenotypes. Mutation of each of these genes results in a physical blockage between the vulva and uterus at the completion of morphogenesis and an egg-laying defective phenotype. The Cog phenotype of egl-26 mutants is a result of adoption of an abnormal morphology by the most apical vulva cell, vulF. EGL-26, a novel protein, is expressed with a distinct subcellular localization pattern around the apical edge of the cell that neighbors vulF called vulE. Thus, we believe that EGL-26 acts non cell-autonomously in controlling cell morphology, and we are currently testing this model. The Cog phenotype in cog-2 mutants is a result of a failure of the anchor cell to be cleared from the apex of the vulva during the fourth larval stage when it normally fuses to the uterine seam cell. COG-2 encodes a Sox ( S RY-related HMG b ox ) domain transcription factor that is expressed in the uterine seam cell. We are conducting a cog-2 suppressor screen in order to identify genes that act together with cog-2 , and we have developed a GFP-based screen to identify additional mutants with phenotypes similar to that of cog-2 mutants.

Awani, Awani et al. (2011) International Worm Meeting "Genome wide RNAi screen to study uv1 cell necrosis."

Awani, Awani, Hanna-Rose, Wendy, Crook, Matt

[International Worm Meeting, 2011]

Necrosis is more than a phenomenon of uncontrolled cell death. There is increasing evidence to suggest that necrosis is genetically programmed. We are exploiting a new model to investigate necrotic cell death. In worms that lack pnc-1 nicotinamidase, the four uv1 cells die by necrosis (1). We want to investigate why uv1 cells die in pnc-1 mutants and to identify genes that promote or inhibit uv1 cell necrosis. In pnc-1 mutant animals with a uv1 cell specific GFP transgene, we can screen for rescue of uv1 cell necrosis by screening for animals that retain the uv1 GFP signal. We aim to screen using the genome-wide RNAi library and expect to gain insight into how and why uv1 cells die in pnc-1 mutants. We are using a split GFP system to engineer animals with uv1 cell specific GFP expression for this screen (2), and we will discuss progress in engineering the strain and preparing for the screen. Reference: 1. EGF signaling overcomes a uterine cell death associated with temporal mis-coordination of organogenesis within the C. elegans egg-laying apparatus. Li Huang , Hanna-Rose Wendy 2. Antiparallel Leucine Zipper-Directed Protein Reassembly: Application to the Green Fluorescent Protein. Indraneel Ghosh , Andrew D. Hamilton , Lynne Regan.

McReynolds, Melanie et al. (2017) International Worm Meeting "Metabolic carbon tracing reveals that disrupted glycolysis arises from a loss of C. elegans salvage NAD+ synthesis."

Hanna-Rose, Wendy, McReynolds, Melanie

[International Worm Meeting, 2017]

As a key link connecting regulatory and bioenergetics process, NAD+ has emerged as a central metabolic co-factor playing a critical role in regulating cellular metabolism and energy homeostasis. Successfully recycling NAM, released from NAD+-consuming reactions, back to NAD+ is a major challenge for maintaining NAD+ metabolism and homeostasis. The first step in C. elegans' salvage NAD+ synthesis involves the nicotinamidase, pnc-1 (T L Vrablik, Huang, Lange, & Hanna-Rose, 2009). Via global metabolic profiling we previously linked loss of salvage NAD+ synthesis, via loss of pnc-1 activity, to disruptions in glycolysis and a subsequent reproductive developmental delay (Wang et al., 2015). However, the citric acid cycle was not perturbed and functions of the mitochondria were intact in pnc-1 mutants (Wang et al., 2015). This observation suggested that loss of salvage NAD+ biosynthesis affects the cytoplasm specifically. To investigate this model, we decided to directly test the effects of loss of pnc-1 on glycolytic and citric acid cycle flux via application of metabolic isotopic carbon tracing tools. After a short four-hour exposure with universally labeled glucose (13C6-Glucose), we were able to detect an isotopically labeled glucose pool in both wild-type animals and pnc-1 mutants. Metabolic carbon tracing confirmed that glycolysis is impeded at the NAD+-dependent step in pnc-1 mutants. Although glycolysis is compromised, we observed no change in the isotopic flow of label from pyruvate to lactate in pnc-1 mutants. Additionally, we observed an increase of isotopic label from pyruvate entering the TCA cycle in these mutants. Although trehalose steady state levels are increased in pnc-1 mutants, we did not observe increased shunting of glucose towards trehalose. This data bolsters the model that loss of NAD+ salvage biosynthesis preferentially affects cytoplasmic functions. Moreover, our work supports stable isotopic labeling as a robust technique to track the passage of metabolites through metabolic pathways in C. elegans.

Moro, Corinna et al. (2021) International Worm Meeting "Tyramine influences associative learning outcomes and is linked to a novel learning phenotype in a purine biosynthesis mutant"

Moro, Corinna, Hanna-Rose, Wendy

[International Worm Meeting, 2021]

Adenylosuccinate lyase deficiency (ASLD; OMIM #103050) is a rare inborn error of purine metabolism disorder. Patients with ASLD have decreased activity of adenylosuccinate lyase (ADSL), which catalyzes two non-sequential steps in the biosynthesis of purines. ASLD is associated with degenerative motor deficits, seizures, mild-to-moderate cognitive impairment, autistic-like behavior, and seizures. We have revealed a novel learning phenotype in C. elegans animals with reduced adenylosuccinate lyase (ADSL-1) activity and are using the model to probe etiology of the neurobehavioral manifestations of ASLD. Naive control animals have a positive chemotactic response to NaCl but change their behavior after starvation (aversive cue) in the presence NaCl (positive cue). After cue-pairing, control animals become indifferent to NaCl in a chemotaxis assay (an associative learning phenotype). adsl-1(RNAi) animals retain the naive chemotactic behavior of control animals, suggesting normal sensory function. However, adsl-1(RNAi) results in an altered learning behavior relative to control animals in response to the cue-pairing. Instead of indifference to NaCl, the adsl-1(RNAi) animals are robustly repelled by NaCl in response to the cue-pairing. We have examined the role of product depletion in this behavioral phenotype via supplementation of cultures with purines and the role of enzyme substrate accumulation using small molecule drugs that inhibit early steps in purine biosynthesis. Both supplementation with adenosine and treatment with lomotrexol effectively revert the learning phenotype in adsl-1(RNAi), suggesting that both effects of reduced ADSL-1 activity are required to manifest the learning phenotype. We used LC-MS to look for clues in the metabolome that might point to how behavior is altered and found that tyrosine levels are elevated in adsl-1(RNAi) animals. Because tyramine and octopamine are produced from tyrosine, we used genetic and pharmacological approaches to probe the role of these neurotransmitter/neuromodulators in associative learning. tdc-1 mutants, which cannot synthesis tyramine, phenocopy adsl-1(RNAi), but tbh-1 mutants, which produce tyramine but not octopamine, do not. Additionally, supplementation of adsl-1(RNAi) animals with tyramine reverts the learning phenotype, suggesting that the behavioral phenotype of adsl-1(RNAi) is caused by a lack of tyramine production. Tyramine is produced by only a few cell types in C. elegans, including the RIM neuron that coordinates reversals during locomotion, providing a link to the behavioral output. We have revealed a novel learning phenotype associated with a purine biosynthesis mutant and are elucidating the metabolic changes in response to adsl-1 depletion that will impact on the downstream effectors identified as important in mediating the behavioral deficiencies.

Crook, Matt et al. (2013) International Worm Meeting "EGF and phosphocholine; a novel mechanism to prevent necrosis."

Hanna-Rose, Wendy, Crook, Matt

[International Worm Meeting, 2013]

Necrosis is not a chaotic uncontrolled reaction to cellular trauma, but a finely controlled cell death program. We have developed a model for analysis of genetic control of necrosis. In our model, nicotinamide induced necrosis of the uterine-vulval uv1 cells in pnc-1 animals is robustly rescued by overactivation of EGF signaling. LET-23, an EGF receptor, has many functions in C. elegans development, and two signaling pathway are known to mediate LET-23 signaling effects; one that signals through LET-60/ Ras and a second through ITR-1. LET-60 signaling downstream of LET-23 is necessary but not sufficient for rescue of necrosis and ITR-1 is neither necessary nor sufficient. These results suggest that an as yet unidentified pathway downstream of LET-23 mediates rescue in concert with the LET-60 pathway. We carried out a targeted RNAi screen and have discovered two genes, pmt-1 and cdk-2, that are required for let-23gf rescue of uv1 necrosis. PMT-1 is a phosphoethanolamine methyltransferase that catalyses the first step of the conversion of ethanolamine to phosphocholine, followed by conversion to phosphatidylcholine. To confirm the relevance of this pathway we knocked down pmt-2, downstream of pmt-1, and supplemented pmt-1 and pmt-2 RNAi animals with choline, an alternative substrate for phosphocholine synthesis. pmt-2 RNAi phenocopied pmt-1 RNAi and choline supplementation restored rescue . However, choline supplementation in a pnc-1 background did not rescue uv1 necrosis, which confirms that phosphocholine synthesis via the PMT pathway is required but not sufficient for rescue. We hypothesised that the PMT pathway is required for let-23gf uv1 rescue to maintain LET-23 cell membrane localization and activity. To test this we investigated the effect of pmt-1 and pmt-2 RNAi on two let-23gf phenotypes; excess vulval induction and excess uv1 cell specification. We found that neither pmt-1 nor pmt-2 RNAi affected either of these phenotypes, which suggests an alternative hypothesis; that phosphocholine itself is required for a LET-23 survival signal. This was supported by the failure of cept-1 or Y49A3A.1 RNAi, both genes required for phosphatidylcholine synthesis, to affect rescue.

McReynolds, Melanie et al. (2015) International Worm Meeting "Cytoplasmic-Specific NAD+ Deficiency Disrupts Glycolysis and Activates Amino Acid Catabolism Affecting Reproductive Development in C. elegans."

Hanna-Rose, Wendy, McReynolds, Melanie

[International Worm Meeting, 2015]

NAD+ is a key metabolite that impacts the entire metabolome because of its role in energy transfer in key metabolic pathways. We previously discovered that NAD+ salvage synthesis through the nicotinamidase PNC-1 is required for normal progression of gonad development in C. elegans. Global metabolic profiling suggested that glycolysis was perturbed in our pnc-1 mutants, which have lower global levels of NAD. We used stable isotope/mass spectrometric flux analysis in wild type and pnc-1 mutants to confirm that glycolysis is compromised when NAD+ salvage synthesis is blocked. In pnc-1 mutants, the turnover rate of universally isotope labeled glucose to phosphoenolpyruvate (PEP) is lower than in wild type, whereas, there was no change in the incorporation of isotope label in dihydroxyacetone (DHAP) between WT and pnc-1 mutants. Although glycolysis is impaired, we had evidence that mitochondrial functions were not changed in pnc-1. Metabolomics analysis showed that unlike other glycolytic intermediates pyruvate levels are not reduced in pnc-1 mutants. Therefore, we hypothesized that excessive use of amino acids as an energy source compensates for insufficient glycolytic flux in pnc-1 mutants. Using a targeted metabolomics approach, we revealed that both alanine and cysteine show a 2-fold increase in our pnc-1 mutants. alpha-ketoglutarate and glutamate levels are also significantly higher in pnc-1 mutants compared to WT. This data suggests that a steady reservoir of amino acids is in place for pyruvate production through protein degradation. We also observed an up-regulation of mRNA expression of important aminotransferase and other enzymes involved in amino acid catabolism in our pnc-1 mutants; supporting the notion that amino acid catabolism is indeed occurring. Our results suggest that compromised glycolysis activates amino acid catabolism to maintain functions of the mitochondria. However, this metabolic shift is not compatible with normal progression of reproductive development in C. elegans. To further investigate this hypothesis, we are extending our stable isotope/mass spectrometric flux experiments to the measurement of amino acid catabolism and are investigating the functional role of amino acid catabolic enzymes using RNAi. .

Crook, Matt et al. (2013) International Worm Meeting "Blocking NAD+ salvage biosynthesis sensitizes specific mechanosensory neurons to nutritional conditions and predisposes them to death."

Crook, Matt, Hanna-Rose, Wendy

[International Worm Meeting, 2013]

While all cells require NAD+ for general metabolism, perturbations in biosynthesis of NAD+ can have surprisingly tissue-specific consequences in physiology and development. We use C. elegans to study the role of NAD+ biosynthetic pathways in cell survival. PNC-1 converts nicotinamide (NAM) to nicotinic acid (NA) in the first step of the invertebrate NAD+ salvage pathway. In pnc-1 mutants, the OLQ neurons, mechanosensory cells involved in head withdrawal and foraging, are sensitized to nutritional conditions and prone to death. OLQ cells die at a low penetrance when grown under normal culture conditions, but OLQ death increases significantly on UV-killed E. coli. Neither accumulation of the PNC-1 substrate NAM nor the lack of PNC-1 product NA is sufficient to cause OLQ death, which suggests that both low NAM and normal NA levels are required for survival. OLQ cells die with a stereotypical progression; the dendrites first bleb, then the cytoplasm swells dramatically in a manner characteristic of a necrotic cell, followed by dendrite and cell body clearance. We sought to compare OLQ death to the well-studied degenerin/ mec-4d-induced touch cell necrosis model. We found that loss of calreticulin function, which suppresses degenerin-induced death, significantly increased OLQ death. Two unc-51/Atg1 alleles, which also suppress degnerin-induced death by blocking autophagy, also increased OLQ death. UNC-51 regulates both autophagy and axon guidance. Knockdown of two other autophagy genes, bec-1 and lgg-1, had no effect. However, mutation of unc-14, an unc-51 axon guidance partner, also showed increased OLQ death, suggesting that correct axon guidance may be essential for OLQ survival. Finally, we found that asp-4(lf) allele, a key effector of necrosis, did not affect OLQ death. These results suggest that OLQ death in pnc-1 animals does not follow a canonical necrosis pathway, yet it is not an apoptotic event, as OLQ death increased in ced-4; pnc-1 double mutants. OLQ death offers a model to probe a novel death pathway in a system with powerful genetic and metabolomic tools at hand.

Chang, Sarah et al. (2015) International Worm Meeting "Mitochondrial sirtuins and oxidative stress response in C. elegans."

Chang, Sarah, Hanna-Rose, Wendy

[International Worm Meeting, 2015]

Oxidative stress, the toxic accumulation of reactive oxygen species (ROS), causes mitochondrial dysfunction, contributing to many age-related diseases such as Alzheimer's, cancer, and diabetes. Mitochondrial sirtuins are conserved proteins involved in mitochondrial function, yet, their specific roles in oxidative stress response are not fully elucidated. It is hypothesized that mitochondrial sirtuins repress ROS by inhibiting key metabolic enzymes from metabolizing nutrients and replenishing the TCA cycle. Preliminary results confirm previous studies showing that sir-2.3 mutants have a shorter lifespan than that of wild-type during oxidative stress, suggesting that mitochondrial sirtuins function during oxidative stress. Additionally, a mutant C. elegans animal missing both its mitochondrial sirtuins, SIR-2.2 and SIR-2.3, is being generating using the CRISPR/Cas9 system. With the double mutant, changes in life span and TCA metabolite levels will be measured. This work helps to understand the function of mitochondrial sirtuins and may lead to novel therapies that combat age-related diseases and oxidative stress-dependent pathologies. .

Estes, Kathleen et al. (2009) International Worm Meeting "A New Role for the Anchor Cell in Vulval Development: Initiating Dorsal Lumen Formation."

Hanna-Rose, Wendy, Estes, Kathleen

[International Worm Meeting, 2009]

Tubulogenesis, the creation of tubular organs during development, is a complicated process involving multiple developmental events including fate specification, cell shape changes and cell-cell interactions. Little is known about initiation of tubulogenesis, particularly how a lumen is established and how two developing tubes connect to form a network. We use the C. elegans vulva and its connection with the uterus to study these complex events. The gonadal anchor cell (AC) functions as the signaling center that induces vulval development, a role that has been well studied. Here we demonstrate that the AC also promotes initiation of lumen formation of the dorsal vulva. In the absence of proper invasion and penetration of the dorsal vulval cells by the AC, the dorsal lumen of the vulva is absent. Using mutants that exhibit aberrant AC invasion of the dorsal vulva, including fos-1, unc-6 netrin and unc-40 netrin receptor, we found that dorsal vulval tubulogenesis is incomplete when the AC fails to invade the vulva. Interestingly, the ventral vulva develops normally in these mutants, suggesting the mechanisms for lumen formation differ between the ventral and dorsal vulva. By examining fate specification and polarity of the dorsal vulval cells, we determined that neither fate specification defects nor polarity defects can account for the aberrant vulF shape. We hypothesize that the absence of a dorsal vulval lumen in these AC invasion mutants is due to lack of the physical presence of the AC within the dorsal vulva as those cells are dividing and undergoing morphogenesis. This demonstrates yet another role of the anchor cell in vulval tubulogenesis and a new mechanism of lumen formation in development.

Shu, Muya et al. (2015) International Worm Meeting "Nicotinamide mononucleotide adenylyltransferase function in C. elegans."

Shu, Muya, Hanna-Rose, Wendy

[International Worm Meeting, 2015]

Nicotinamide adenine dinucleotide (NAD+) is an essential metabolite in the cell. It serves as a cofactor as well as a substrate for various enzymes and participates in signaling pathways. There are two NAD+ pools in cells: nuclear/cytoplasmic and mitochondrial. These pools are separately maintained and have different metabolic roles. Our lab has shown that a reduction in NAD+ bioavailability contributes to gonad development in C. elegans. We want to determine how the subcellular location of NAD+ formation affects gonad development and to investigate the function of the distinct pools of NAD+. Nicotinamide mononucleotide adenylyltransferases (NMATs) convert nicotinamide mononucleotide to NAD+ or nicotinic acid mononucleotide to nicotinic acid adenine dinucleotide. In humans, there are three NMNAT genes. Nuclear NAD+ is synthesized by NMNAT1. NMNAT2 docks in Golgi, and NMNAT3 is the mitochondrial enzyme that sustains the mitochondrial NAD+ pool. We would like to use NMNAT function to explore the compartment specific requirements for NAD+ biosynthesis in C. elegans gonad development. However, it is unclear which of the two C. elegans NMNAT genes, nmat-1 and nmat-2, corresponds to which human gene.nmat-1 and nmat-2 are both listed as NMAT3 homologs in Wormbase. However, our own phylogenetic analysis failed to substantiate this relationship and did not clarify orthologous relationships. We hypothesize that one of the genes is for nucleocytoplasmic NAD+ biosynthesis and the other is for mitochondrial biosynthesis. To being to investigate this hypothesis, we have created translational fusions of nmat-1 and nmat-2 to GFP under the control of the myo-3 promoter and examined their subcellular localization in muscle cells. NMAT-1 is localized in mitochondria, and NMAT-2 is localized in the cytoplasm. We have also examined nmat-2 functionally. nmat-2(tm2905) animals have a gonad development phenotype, suggesting the cytoplasmic pool of NAD+ is critical for gonad development. This result is consistent with other experiments in the lab pointing to a relationship between cytoplasmic NAD+ and gonad development. We have used the CRISPR/Cas9 system to create an allele of nmat-1. The nature of the allele is being analyzed, and we expect to present results at the meeting. The data from these experiments will help us to understand the relationship between the nuclear/cytoplasmic and mitochondrial NAD+ pools in terms of gonad development and will be a useful tool to probe other functions of NAD+ biosynthesis as well.