Warnhoff, Kurt et al. (2009) International Worm Meeting "Segmenting Early Embryogenesis in Caenorhabditis elegans."

Wehrman, Alexandra, Thatcher, Scott, Warnhoff, Kurt, Walston, Timothy

[International Worm Meeting, 2009]

An understanding of the forces acting on cells in the early embryo can provide important information for how cells interact to determine their shapes, movements and fates. These forces can be physical and genetic, as well as intracellular or extracellular. The early Caenorhabditis elegans embryo provides an excellent environment to explore the forces acting during embryogenesis and to develop techniques and models that can be applied later to more advanced biological events. We report the continued development of a four-dimensional GGH (Glazier-Granier-Hogeweg) model to simulate the four-cell stage of embryogenesis. In addition, a semi-automated procedure for the segmentation of four-dimensional differential interference contrast (DIC) microscopic data has been developed. A precise and accurate segmentation scheme offers many opportunities for the quantitative analysis of early embryogenesis and other cellular systems. Our segmentation technique incorporates a combination of level set and watershed methods to properly delineate cells of the embryo in DIC micrographs. Segmented data serves as an input into the current GGH code and data from many embryos are needed as a source of calibration and validation for the model. This segmentation technique for DIC images of the C. elegans embryo should also be applicable to DIC images of other embryos or biological samples.

Warnhoff, Kurt et al. (2021) International Worm Meeting "Interkingdom transfer of molybdenum cofactor from bacteria to C. elegans"

Hercher, Thomas, Warnhoff, Kurt, Ruvkun, Gary, Mendel, Ralf

[International Worm Meeting, 2021]

The molybdenum cofactor (Moco) is a 520 dalton prosthetic group that is synthesized in a multi-step enzymatic pathway present in Archaea, Bacteria, and Eukarya. In animals, four oxidases (among them sulfite oxidase) use Moco as a prosthetic group. Moco is essential in animals; humans with mutations in genes that encode Moco-biosynthetic enzymes display lethal neurological and developmental defects. Moco supplementation seems a logical therapy, however free Moco is too fragile to be purified and administered therapeutically. Surprisingly, Moco biosynthesis is not essential in the nematode Caenorhabditis elegans if they are fed bacteria that synthesize Moco. Our genetic and biochemical studies demonstrate that protein-bound Moco is the stable, bioavailable species of Moco taken up by C. elegans from bacteria and is an effective dietary supplement in a C. elegans model of Moco deficiency. These findings establish an animal pathway for Moco transport and suggest a novel therapeutic strategy for Moco deficiency.

Warnhoff, Kurt et al. (2013) International Worm Meeting "natc-1 mediates stress resistance and dauer formation as a downstream effector of the insulin/IGF-1 signaling pathway."

Murphy, John T., Peterson, Michelle, Hsu, Simon, Kornfeld, Kerry, Schneider, Daniel L., Warnhoff, Kurt

[International Worm Meeting, 2013]

Animals are exposed to a wide variety of environmental stresses, and they may employ both specific and general strategies to tolerate such stresses. To identify mechanisms that permit C. elegans to tolerate high levels of dietary zinc, we screened for mutations that enhanced growth and survival in high levels of supplemental zinc. We identified a C. elegans N-terminal acetyltransferase (NAT) gene in this screen, which we named natc-1. natc-1 encodes a protein that is homologous to the vertebrate NAA35, an auxiliary subunit of the NatC complex. NATs function in complexes to catalyze the N-terminal acetylation of proteins, which is a widespread modification that affects the majority of eukaryotic proteins. However, relatively little is known about the biological functions of NATs. We demonstrated that mutations in natc-1 cause resistance to a broad spectrum of physiologic stressors, including heavy metals, heat, and oxidation. natc-1 was identified based on resistance to zinc excess, yet the stress resistance conferred by natc-1 loss-of-function is protective against a wide range of environmental stresses. The C. elegans FOXO transcription factor DAF-16 is a key component of the insulin/IGF-1 pathway that mediates general stress resistance and has been predicted to directly bind the natc-1 promoter. To characterize the role natc-1 plays in insulin/IGF-1 signaling, we analyzed molecular and genetic interactions with key components of the insulin/IGF-1 pathway. We demonstrated that natc-1 is transcriptionally repressed by DAF-16 and that mutations in natc-1 are epistatic to daf-16 mutations with respect to stress resistance. Furthermore, natc-1 inhibits dauer formation in a sensitized genetic background. Based on these findings, we hypothesize that natc-1 functions as a downstream effector of the insulin/IGF-1 signaling pathway to mediate stress resistance and dauer formation. These studies identify a novel biological function for natc-1 as a modulator stress resistance and dauer formation and define a functionally significant downstream effector of the insulin/IGF-1 signaling pathway.

Warnhoff, Kurt et al. (2015) International Worm Meeting "nhr-33 encodes a nuclear receptor that is regulated by high zinc and mediates transcriptional activation to maintain zinc homeostasis in C. elegans."

Kornfeld, Kerry, Tan, Chieh-Hsiang, Schneider, Daniel, Roh, Hyun, Kocsisova, Zuzana, Croswell, Damari, Warnhoff, Kurt, Morrison, Andrew

[International Worm Meeting, 2015]

Zinc is an essential element involved in many biological processes and human disorders. Excess zinc is deleterious, and therefore animals have evolved sophisticated mechanisms to maintain zinc homeostasis in response to variable zinc availability. However, mechanisms of zinc sensing and detoxification are not well defined. Caenorhabditis elegans responds to excess dietary zinc by activating transcription of critical zinc-homeostasis genes, such as the zinc exporters cdf-2 and ttm-1b. We demonstrated that the transcriptional response to high dietary zinc is mediated by the high zinc activated (HZA) element, a 14 bp DNA enhancer that is present in multiple genes that are induced by high zinc (Roh et al., NAR, 2014). To identify genes that are necessary for the response to excess dietary zinc, we conducted a forward mutagenic screen for animals with defects in zinc-induced transcription. We identified five loss-of-function mutations and one gain-of-function mutation in nhr-33, which encodes an uncharacterized nuclear receptor. Nuclear receptors are transcription factors that have a DNA binding domain and a ligand binding domain. nhr-33(lf) mutations abrogate the induction of zinc-regulated genes and dramatically impair zinc homeostasis, as documented by reduced growth and development when mutant animals were cultured with excess dietary zinc. By contrast the nhr-33(gf) mutation caused constitutive induction of zinc-regulated genes. Thus, nhr-33 is necessary and sufficient for high zinc-activated transcription. To characterize the mechanism of action of nhr-33, we analyzed the expression pattern; NHR-33 is expressed in intestinal cells and accumulates in nuclei in response to high dietary zinc. Biochemical experiments demonstrated that NHR-33 binds the HZA element with high specificity. These results indicate that nhr-33 is the master regulator of high zinc homeostasis in C. elegans. High dietary zinc causes NHR-33 to accumulate in the nucleus where it binds the HZA element and activates transcription of genes that promote zinc homeostasis. These findings establish a new paradigm for environmental sensing by identifying a novel and unexpected role for a nuclear receptor in metal homeostasis.

Snoozy, Jennifer et al. (2022) MicroPubl Biol

Snoozy, Jennifer, Ruvkun, Gary, Warnhoff, Kurt, Breen, Peter C

[MicroPubl Biol, 2022]

Molybdenum cofactor (Moco) is an essential prosthetic group that mediates the activity of 4 animal oxidases and is required for viability. Humans with mutations in the genes encoding Moco-biosynthetic enzymes suffer from Moco deficiency, a neonatal lethal inborn error of metabolism. Caenorhabditis elegans has recently emerged as a useful and tractable genetic discovery engine for Moco biology. Here, we identify and characterize K10D2.7/moc-6, the C. elegans ortholog of human MOCS2A, a sulfur-carrier protein essential for Moco synthesis. Using CRISPR/Cas9 gene editing, we generate 3 null mutations in K10D2.7/moc-6 and with these alleles genetically demonstrate that K10D2.7/moc-6 is necessary for endogenous Moco synthesis in C. elegans.

Dietrich, Nicholas et al. (2015) International Worm Meeting "Identification of an enhancer element that mediates transcriptional activation in response to low dietary zinc."

Roh, Hyun, Kornfeld, Kerry, Schneider, Daniel, Warnhoff, Kurt, Dietrich, Nicholas

[International Worm Meeting, 2015]

Zinc is a trace element that is essential for the structure and function of a wide range of proteins, and disrupted zinc metabolism is implicated in a number of human diseases. Because both zinc deficiency and excess are deleterious, animals require homeostatic mechanisms to respond to dietary changes in zinc availability. In biological systems, zinc exists as a divalent cation that cannot passively cross cell membranes. Therefore, transmembrane proteins that transport zinc across the plasma membrane and the membranes of intracellular organelles play a critical role in zinc trafficking. Two families of evolutionarily conserved zinc transporters have been characterized in animals. Cation diffusion facilitator (CDF) proteins reduce the level of cytoplasmic zinc by transport out of cells or into the lumen of intracellular organelles, whereas Zrt, Irt-like proteins (ZIPs) increase the level of cytoplasmic zinc by transport in the opposite direction. C. elegans has been used to study the function of CDF proteins, but little work has been done to characterize ZIP proteins. To analyze the regulation of ZIP genes, we used qRT-PCR to analyze transcript levels in response to low dietary zinc. Three ZIP genes displayed increased transcript levels in response to zinc deficiency. We used bioinformatics to identify a DNA motif that is present in the promoters of all three regulated genes, which we named the low zinc activated (LZA) element. The LZA was necessary for low zinc activated transcription in native promoters and it was sufficient to mediate low zinc activated transcription in a basal promoter. To characterize the function of ZIP genes, we used feeding RNAi to analyze growth and development in low dietary zinc induced with a zinc chelator. RNAi against Y54G9A.4 caused sensitivity to low zinc. This phenotype was also observed in animals with a deletion allele. Thus, Y54G9A.4 is necessary for survival and growth in low dietary zinc, suggesting it plays an important role in zinc homeostasis. Using transgenic animals, we demonstrated that Y54G9A.4 is expressed in intestinal cells, and the promoter of this gene is induced by low dietary zinc. These studies identify important factors in the response to low zinc including an enhancer element that mediates transcriptional activation and a ZIP transporter that promotes homeostasis. .

Tan, Chieh-Hsiang et al. (2015) International Worm Meeting "hke-4.1/ ZIP7 encodes a zinc transporter involved in zinc homeostasis and sperm function in Caenorhabditis elegans."

Dietrich, Nicholas, Kornfeld, Kerry, Warnhoff, Kurt, Ellis, Ronald E., Tan, Chieh-Hsiang, Zhao, Yanmei

[International Worm Meeting, 2015]

Zinc is an essential trace element that plays many important roles in biological systems. The homeostasis of zinc in eukaryotes is mediated by two conserved families of transmembrane proteins- Znt/CDF (Cation Diffusion Facilitator) proteins transport zinc out of the cytoplasm, whereas ZIP (Zrt/Irt-like protein) proteins transport zinc into the cytoplasm. Based on the existence of CDF and ZIP homologues for every zinc transporter in humans, the nematode Caenorhabditis elegans is a promising model for the study of zinc homeostasis. While important progress has been made in understanding CDF proteins in worms, there is very little information about the function of ZIP proteins. Studies of human cells suggest that ZIP7 functions as a key component of intracellular zinc signaling by mediating release of ER-stored zinc molecules after being activated by phosphorylation. There are two homologues of human ZIP7 in C. elegans, hke-4.1 and hke-4.2. To investigate the function of hke-4.1, we analyzed a deletion mutation that creates a null allele. Loss of hke-4.1 caused worms to be hypersensitive to low dietary zinc induced by using a zinc chelator. Moreover, the level of hke-4.1 mRNA was increased by low dietary zinc. Thus, hke-4.1 is induced by low dietary zinc and necessary for growth in these conditions, suggesting it plays a role in zinc trafficking and homeostasis. Loss of hke-4.1 also caused a partially penetrant, cold-sensitive sterile phenotype in hermaphrodites and reduced the fertility of males. The sterile phenotype of the hermaphrodites was rescued by mating to wild-type males. These results indicate hke-4.1 is necessary for sperm function. We are currently analyzing the sperm defects in hke-4.1 mutant animals and investigating the hypothesis that hke-4.1 mediates a zinc signal during C. elegans sperm development.

Sewell AK et al. (2021) Genes Dev "Learning from the worm: the effectiveness of protein-bound Moco to treat Moco deficiency."

Sewell AK, Han M

[Genes Dev, 2021]

Molybdenum cofactor (Moco) is synthesized endogenously in humans and is essential for human development. Supplementation of Moco or its precursors has been explored as a therapy to treat Moco-deficient patients but with significant limitations. By using the nematode <i>C. elegans</i> as a model, Warnhoff and colleagues (pp. 212-217) describe the beneficial impact of protein-bound Moco supplementation to treat Moco deficiency. If such an effect is conserved, this advance from basic research in worms may have significant clinical implications as a novel therapy for molybdenum cofactor deficiency.

Roh, Hyun Cheol et al. (2013) International Worm Meeting "Identification of cis-regulatory elements that confer zinc-responsive transcription in intestinal cells of C. elegans."

Zhao, Guoyan, Warnhoff, Kurt, Roh, Hyun Cheol, Dimitrov, Ivan, Deshmukh, Krupa, Tsai, Wendy, Cabrera, Daniel, Kornfeld, Kerry

[International Worm Meeting, 2013]

Zinc is an essential metal involved in a broad range of biological processes; it serves as a structural and/or enzymatic cofactor in many proteins and as a signaling molecule. Because both zinc deficiency and excess adversely affect the health of humans and other animals, sophisticated mechanisms have evolved to mediate zinc homeostasis. One important homeostatic mechanism is regulation of transcription in response to fluctuations of dietary zinc. To use C. elegans to characterize zinc-regulated transcription, we identified four genes that are involved in zinc metabolism and displayed transcriptional induction by dietary zinc; mtl-1 and mtl-2 encode metallothionein proteins that are predicted to bind and sequester zinc ions, and cdf-2 and ttm-1b encode CDF family zinc transporters. Using a bioinformatic motif search, we identified a conserved DNA element in these four zinc-responsive genes, which we named the C. elegans zinc-responsive element (ZRE). The ZRE was conserved in other nematode species and also present in previously described cadmium-responsive genes. Mutagenesis studies in transgenic animals showed that the ZRE was necessary for zinc-mediated transcriptional induction in intestinal cells. Furthermore, the motif was sufficient to confer zinc responsiveness on a basal promoter. ELT-2 is a transcription factor that is critical for intestinal development, and we noticed that the ZREs were closely associated with a predicted binding site for ELT-2. We demonstrated that the predicted ELT-2 binding site was necessary in cis and the ELT-2 transcription factor was necessary in trans for zinc-mediated transcriptional induction. These findings suggest that the coordinated activity of the ZRE and the ELT-2 binding site mediate zinc-responsive transcriptional activation in intestinal cells. We are using this signature of a ZRE and an ELT-2 binding site to computationally predict novel zinc-responsive genes. These studies advance our understanding of molecular mechanisms of zinc-responsive transcriptional regulation and contribute to understanding zinc metabolism and homeostasis.

Earley, Brian et al. (2021) International Worm Meeting "Cadmium hijacks the high zinc response by binding and activating the HIZR-1 nuclear receptor"

Ahmad, Raheel, Earley, Brian, Kornfeld, Kerry, Schneider, Daniel, Warnhoff, Kurt, Alcantar, Alan, Cubillas, Ciro, Lyon, Maximilian

[International Worm Meeting, 2021]

Cadmium is an environmental pollutant and significant health hazard that is similar to the physiological metal zinc. In C. elegans high zinc homeostasis is regulated by the HIZR-1 nuclear receptor transcription factor. To define relationships between high zinc homeostasis and the response to cadmium, we identified 145 cadmium-regulated genes. hizr-1 was necessary for activation of a subset of genes, indicating there are at least two mechanisms of cadmium-regulated transcription. Cadmium directly bound HIZR-1, promoted nuclear accumulation of HIZR-1 in intestinal cells, and activated HIZR-1-mediated transcription via the HZA enhancer. Thus, cadmium binding promotes HIZR-1 activity, indicating that cadmium acts as a zinc mimetic to hijack the high zinc response. To elucidate the relationships between high zinc and cadmium detoxification, we analyzed three pathways: the phytochelatin pathway strongly promoted cadmium resistance but not high zinc resistance, the hizr-1 pathway strongly promoted high zinc resistance but not cadmium resistance, and the mek-1/sek-1 pathway promoted resistance to high zinc and cadmium. These studies identify resistance pathways that are specific for high zinc and cadmium as well as a shared pathway.