Juozaityte, Vaida et al. (2011) International Worm Meeting "Searching for factors required for BAG cell fate determination."

Pocock, Roger, Juozaityte, Vaida

[International Worm Meeting, 2011]

The BAG neurons enable C. elegans to generate rapid behavioral responses to oxygen and carbon dioxide. However, the molecular factors that determine BAG cell fate are not known. The aim of this project is to identify the molecular factors required for the differentiation of BAG neurons. First, we aim to identify elements in the promoters of genes expressed in the BAG neurons that are required for BAG expression. We are systematically dissecting the promoters of genes expressed in the BAG neurons - flp-11, flp-13, flp-17, flp-19, gcy-31 and gcy-33. Such deletion analysis will enable the identification of minimal elements within these promoters that are required for BAG expression. Second, we will search for putative transcription factor binding sites within these minimal elements that are required for BAG expression. We will subsequently use BAG::GFP reporter strains to examine BAG cell fate in relevant transcription factor mutant strains. The results of the project will enable us to better understand how the specification of BAG neurons is regulated in C. elegans and potentially identify crucial factors required for oxygen and carbon dioxide sensing in higher organisms.

Ringstad, Niels et al. (2009) International Worm Meeting "The BAG Sensory Neurons are Activated by Environmental Carbon Dioxide."

Hallem, Elissa, Horvitz, Bob, Ringstad, Niels, Sternberg, Paul

[International Worm Meeting, 2009]

The BAG neurons are ciliated sensory neurons that recently have been shown to mediate avoidance behavior to carbon dioxide (CO2) and to inhibit egg-laying behavior. To test whether the BAG neurons are CO2 sensors, we generated transgenic animals expressing the high-affinity genetically encoded calcium sensor cameleon YC3.60 in the BAG neurons. We observed increases in BAG neuron calcium in response to application of as little as 0.1% CO2 with half maximal increases evoked by application of 0.9% CO2. The BAG neurons require the TAX-2 / TAX-4 cyclic nucleotide-gated cation channel to respond to environmental CO2. The BAG neurons of rgs-3 mutants, which lack a negative regulator of heterotrimeric G proteins, are defective for CO2-evoked calcium responses, indicating that the BAG neurons are negatively regulated by a G protein signaling pathway. Using in vivo calcium imaging, we are seeking neurons that function downstream of the BAG sensory neurons in neuronal circuits that mediate CO2 avoidance and the regulation of egg laying.

Choi, Jung-Hwan et al. (2013) International Worm Meeting "Redundant mechanisms for modulation of the serotonergic HSNs by an environmental cue."

Choi, Jung-Hwan, Ringstad, Niels

[International Worm Meeting, 2013]

Egg-laying in worms is regulated by hermaphrodite-specific neurons (HSNs), which stimulate the vulval muscles through serotonin. Previous studies have shown that cholinergic and peptidergic input to HSNs inhibits egg-laying. However, it has been shown that removal of only one of these inhibitory inputs to HSNs cannot induce egg-laying, but removal of both inputs induces precocious egg-laying. BAG neurons have been shown to the primary source of HSN inhibition through the neuropeptide, FLP-17, and its receptor, EGL-6, a G protein-coupled receptor expressed in HSNs. BAG-ablated wildtype animals show normal egg-laying behavior, whereas BAG-ablated unc-17 mutants, which lack the vesicular acetylcholine transporter, have a precocious egg-laying phenotype. This suggests there are redundant inhibitory pathways, one that requires BAG neurons and FLP-17 and another that requires cholinergic transmission. To better understand how the BAG-dependent and BAG-independent pathways contribute to HSN inhibition in vivo, we propose to perform calcium imaging of HSNs in response to BAG neuron stimulation in wildtype and mutant animals which lack cholinergic or peptidergic input.

Cornils, Astrid et al. (2011) International Worm Meeting "Mechanisms of BAG sensory cilia specification."

Sengupta, Piali, Cornils, Astrid

[International Worm Meeting, 2011]

The nervous system relies on specialized sensory neurons to sense environmental conditions and initiate appropriate physiological and behavioral responses. Functions of many sensory neurons are dependent on the presence of morphologically specialized cilia which are microtubule-based organelles acting as cellular 'antennae'. A mechanism called intraflagellar transport (IFT) is required to build cilia and transport cargo and ciliary precursors into the ciliary compartment. However, little is known about how diverse, specialized sensory cilia are shaped. Caenorhabditis elegans is an established model for the study of cilia generation and maintenance. Worms possess 60 ciliated neurons, each of which is specialized to sense specific cues. The BAGL/R sensory neurons, so-called due to the bag-like appearance of their cilia, sense carbon dioxide and decreases in oxygen levels. Little is known about the mechanisms by which BAG cilia are formed, although interestingly, the essential IFT component osm-6/IFT52 has been reported not to be expressed in the BAG neurons. To investigate the formation of BAG cilia, I am first analyzing BAG cilia structure in known ciliary mutant backgrounds to determine the extent to which BAG ciliogenesis depends on known ciliary genes. I am analyzing in vivo IFT as well as the ultrastructure of BAG cilia in wild-type animals, and plan to carry out a forward genetic screen to identify new genes required for BAG ciliogenesis. Comparing the pathways for BAG cilia specialization to those of other specialized C. elegans neuronal cilia will allow me to define new mechanisms for the generation of ciliary structure diversity.

Garrett, Destane et al. (2017) International Worm Meeting "The transcription factor fkh-8 is involved in sustaining CO2 function in C. elegans."

Nelms, Brian, Garrett, Destane, Cawthon, Bryan

[International Worm Meeting, 2017]

Sensing and responding to changing levels of oxygen and carbon dioxide is an important function across the animal kingdom. In the model organism Caenorhabditis elegans, the inability to sense fluctuations in CO2 levels can potentially lead to death. The BAG neuron is one cell type that can function as a mediator of high CO2 avoidance and can sense decreases in O2 levels. Some transcription factors, such as ETS-5 and EGL-13, are already known to be required for BAG neuron CO2 and O2 sensing. We have observed through genetics and behavioral studies that the transcription factor FKH-8 likely also plays a role in CO2 sensing. We are more specifically studying the role of FKH-8 in regulating BAG neuron gene expression by examining expression decreases or increases in specific BAG neuron reporter genes in fkh-8 mutants. Through these experiments, we hope to identify which important signaling components in BAG neuron CO2-sensing are controlled by FKH-8.

Handley, A et al. (2019) International Worm Meeting "ETS-5 regulates BAG-specific insulin signalling to control intestinal metabolism."

Handley, A, Wu, Q, Pocock, R, Sherry, T

[International Worm Meeting, 2019]

Metabolic homeostasis is essential to life. The nervous system is the master metabolic coordinator that integrates input information such as external nutrient availability, levels of internal nutrient stores, and even past experiences. The outputs from the nervous system include changing food consumption, storing excess energy and nutrients, deciding what that excess should be stored as, and ultimately altering behaviour. Using Caenorhabditis elegans as a model - we investigate how gene-regulatory mechanisms in the nervous system control organismal metabolism and behaviour. We recently discovered that the conserved transcription factor ETS-5/Pet1, acts from the BAG and ASG neurons to control intestinal fat levels and exploration behaviour. Precisely how ETS-5 functions in the nervous system to affect intestinal fat levels is unknown. However, we have shown that neuropeptide secretion from the BAG neurons is an important factor in this pathway. We hypothesised that ETS-5 transcriptionally regulates neuropeptide expression in the BAG neurons, and that these BAG-specific neuropeptides in turn regulate intestinal metabolism. To test this hypothesis, we screened BAG-expressed neuropeptides for exploration defects and identified INS-1. Although INS-1 is expressed in multiple neurons, we show that INS-1 expressed specifically in the BAG neurons regulates intestinal fat levels and exploration behaviour. Our subsequent analysis revealed that ETS-5 directly binds to, and regulates the ins-1 promoter in the BAG neurons. This regulatory mechanism prevents fat storage and promotes exploration. Intriguingly, we found that excess nutrients in the intestine can reduce both ETS-5 and INS-1 levels within the BAG neurons, revealing a nutrient-dependent feedback loop. Together, this work reveals a neuron-intestinal signalling circuit that is critical for maintaining metabolic homeostasis.

Beverly, Matthew H. et al. (2009) International Worm Meeting "The BAG neurons respond to temperature, and are necessary for cryophilic behavior."

Beverly, Matthew H., Sengupta, Piali

[International Worm Meeting, 2009]

C. elegans exhibits complex behavioral responses to thermal stimuli. When placed on a spatial thermal gradient at temperatures above its cultivation temperature (Tc), worms will move down the gradient towards colder temperatures (cryophilic behavior), whereas at temperatures below the Tc, worms are atactic. At temperatures around the Tc, worms track isotherms. The underlying circuit required for these behaviors includes the AFD sensory neurons, as well as the AIY and AIZ interneurons. A current model of circuit function in thermosensory navigation behavior suggests that there is an underlying default cryophilic drive regulated by the AIZ interneurons and as yet unknown sensory neurons. Exhibition of the cryophilic drive is restricted to temperatures above the Tc via activity of the AFD and AIY neurons, which are also essential for isothermal tracking behavior. The goal of my project is to identify additional components of the thermosensory circuit, and in particular, to identify the sensory neuron(s) mediating cryophilic behavior. We found that animals with genetically ablated BAG neurons exhibit strong defects in cryophilic behavior. Although the BAG neurons are not directly presynaptic to the AIZ interneurons required for cryophilic behavior, this neuron type is presynaptic to the RIR and RIG interneurons which in turn synapse directly onto AIZ. The BAG neurons have previously been implicated in oxygen and carbon dioxide sensation; however, our experiments suggest that temperature responses are independent of gas sensation in this neuron type. BAG expresses known genes involved in sensory pathways including the tax-2/4 cyclic nucleotide gated ion channels as well as the soluble guanylyl cyclases gcy-31 and -33, gcy-31 and -33 mutants also show defects in cryophilic behavior on a thermal gradient. Preliminary calcium imaging data indicate that BAG responds to both small increases and decreases in temperature (0.2 degC) above and below the animal''s cultivation temperature. I am further characterizing the responses of BAG to temperature by examining changes in intracellular calcium and cGMP levels using genetically encoded sensors. I will also determine whether communication between the AFD and BAG neurons play a role in mediating temperature responses, and identify additional BAG-expressed genes required for thermosensory signal transduction.

Smith, Ewan St. John et al. (2013) International Worm Meeting "Chemical tuning of CO2-responsive BAG neurons."

Ringstad, Niels, Smith, Ewan St. John, Martinez-Velazquez, Luis Antonio

[International Worm Meeting, 2013]

Animals from diverse phyla possess neurons that are activated by the product of aerobic respiration, carbon dioxide (CO2). It has long been thought that such neurons primarily detect the products of CO2 hydration, protons and bicarbonate. To identify the specific chemical cue that activates BAG neurons, we studied isolated BAG neurons in culture, using methods that allow both monitoring of cell physiology and control of the extracellular and intracellular environments. We show that the majority of isolated BAG neurons showed calcium responses to molecular CO2, although a fraction of these cells can also be activated by acid stimuli. These responses to acid stimuli were not seen when the BAG neurons were tested in situ. One component of the BAG transduction pathway, the receptor-type guanylate cyclase GCY-9, suffices to confer cellular sensitivity to both CO2 and acid, indicating that it is a bifunctional chemoreceptor. We speculate that in other animals, receptors similarly capable of detecting molecular CO2 might mediate effects of CO2 on neural circuits and behavior.

Redo Riveiro, Alba et al. (2013) International Worm Meeting "egl-46 is a novel BAG cell fate modulator."

Pocock, Roger, Redo Riveiro, Alba

[International Worm Meeting, 2013]

Genetic studies in C. elegans attempt to identify molecular components required for neural circuits to function correctly. This study focuses on BAG neurons, which have the major role in oxygen (O2) and carbon dioxide (CO2) sensing. Development of BAG neurons is controlled by the conserved transcription factors ETS-5 (Brandt, Aziz-Zaman et al. 2012) and EGL-13 (Petersen et al. 2013-in press). However the effect of ets-5 and egl-13 loss of function mutants does not affect all BAG terminal fate markers. Therefore, other genes must act in parallel to these factors to specify BAG fate. In a classical forward genetic screen, we identified egl-46 as a BAG fate modulator. EGL-46 is a zinc finger transcription factor that regulates terminal cell divisions in the Q lineage where egl-46 mutants undergo extra rounds of terminal Q cell divisions (Wu, Duggan et al. 2001). The egl-46 mammalian ortholog Insm1 has a similar function in determining cell fates. Insm1 has been implicated in the development of pancreas (Farkas, Haffner et al. 2008), cortex and hindbrain (Jacob, Storm et al. 2009). Insm1 mutant mice present a thicker layer of proliferative progenitors and a thinner neuro-basal layer in the olfactory ephitelium (Rosenbaum, Duggan et al. 2011). Here we show that egl-46 mutants have defects in the expression of specific terminal markers in the BAG neurons. We have rescued these defects by transgenically expressing a fosmid containing the egl-46 locus. Currently, we are performing genetic interaction studies with BAG-fate regulators like ets-5 and egl-13.

Gang, Spencer et al. (2015) International Worm Meeting "Host-seeking strategies of skin-penetrating parasitic nematodes."

Tselikova, Anastassia, Okubo, Ryo, Hallem, Elissa, Castelletto, Michelle, Gang, Spencer, Barnia, Arezoo

[International Worm Meeting, 2015]

Skin-penetrating nematodes such as the threadworm Strongyloides stercoralis and the hookworms Ancylostoma duodenale and Necator Americanus infect approximately 1 billion people worldwide and pose dangerous health risks, including severe intestinal distress, stunted growth and cognitive impairment in children, and heightened susceptibility to other infectious diseases. The infective juveniles (IJs) of skin-penetrating nematodes are thought to search for hosts using sensory cues, but their host-seeking behavior is poorly understood. To understand host-seeking behavior in more detail, we asked how S. stercoralis moves in the absence of chemosensory stimuli. We found that S. stercoralis is highly motile and uses a cruising strategy to search for potential hosts. We next asked how increased temperature might alter host-seeking behavior. We found that at 37degC S. stercoralis shows increased speed and engages in local search behavior. S. stercoralis is also attracted to human skin and sweat odorants, many of which are known mosquito attractants. We tested olfactory preferences at different life stages and found that the free-living stages are highly attracted to host feces while IJs are not, suggesting a mechanism by which host seeking is restricted to IJs. We compared host odorant responses of S. stercoralis to six other nematode species and found that parasite olfactory preferences reflect host specificity rather than phylogeny, suggesting an important role for olfaction in host selection. We are now examining the neural basis of host-seeking behavior. We focused initially on the neural basis of CO2 response. In C. elegans, the receptor guanylate cyclase GCY-9 is required for CO2 detection by BAG neurons. We identified a gcy-9 homolog in S. stercoralis and generated a gcy9::GFP construct, which specifically labeled putative BAG neurons based on anatomical position. We then generated a gcy9::GCaMP3 construct and imaged CO2-evoked neural activity in BAG neurons of S. stercoralis infective juveniles. We found that S. stercoralis and C. elegans BAG neurons respond similarly to CO2, suggesting the neural basis of CO2 detection is conserved across species. We are now employing a similar reporter-based strategy to identify and image from S. stercoralis olfactory neurons in response to host-emitted odorants. To identify molecular components required for host seeking behavior, we are developing CRISPR-Cas9 mediated mutagenesis in S. stercoralis. Understanding how skin-penetrating nematodes target human hosts could lead to new strategies for preventing infections. .