Singhvi, Aakanksha et al. (2013) International Worm Meeting "Regulation of sensory neuron architecture."

Singhvi, Aakanksha, Shaham, Shai, Friedman, Christine

[International Worm Meeting, 2013]

The shapes of neuronal receptive endings are exquisitely tuned to their function. Receptive endings of some sensory cells, including photoreceptors and auditory hair cells, have microvilli that allow dense packaging of receptors in a small space. To understand how sensory neurons form and maintain microvilli, we are studying the receptive endings of the C. elegans AFD thermosensory neuron, which are enveloped by the AMsh glia. Using cell ablations and inducible inhibition of secretion, we demonstrated that glia-secreted cues dynamically regulate the shape of AFD microvilli. To identify these glial signals, we performed a forward genetic screen seeking mutants with defects in AFD shape. Of the six mutants identified, we have cloned four. One mutant affects the gene unc-23, encoding a BAG2 co-chaperone that functions with Hsp70 to control protein folding. Microvilli defects in these mutants are progressive and coincident with the appearance of large vesicular structures within glia, suggestive of a secretory block. Intriguingly, mutations in human Hsp genes underlie a subset of sensory neuropathies characterized by progressive glial myelin damage and axonal tip degeneration. Our analyses lead us to propose a model where UNC-23 regulates protein folding of a small number of polypeptides in glia by antagonizing the E3/E4 ubiquitin ligase, CHIP/CHN-1. A second mutant we isolated affects the gene gcy-8, encoding an AFD-expressed receptor guanylyl cyclase important for thermosensation. Our analyses suggest that deregulated cyclase activity in AFD alters microvilli. Strikingly, receptor guanylyl cyclase mutations result in structurally abnormal microvilli in rod and cone cells of the eye. Finally, our preliminary data also suggest a role for the engulfment pathway in maintaining AFD microvilli. This is intriguing, given that engulfment by retinal pigmented epithelial cells sculpts rod and cone microvilli in mammals. Taken together, our studies provide new insight into how glia-neuron communication controls neuronal receptive ending morphology, and suggest that mechanisms governing receptive ending shape may be conserved.

Singhvi, Aakanksha et al. (2015) International Worm Meeting "Glia engulf AFD neuron receptive endings."

Bae, Andrea, Shaham, Shai, Singhvi, Aakanksha

[International Worm Meeting, 2015]

Glia participate in pruning of neuronal receptive endings during development and in response to plastic changes in the nervous system. We present evidence that glial engulfment of neuronal receptive endings also takes place in C. elegans. We find that animals expressing GFP targeted to the sensory receptive ending of the AFD sensory neuron also display GFP fluorescence in punctate structures within the amphid sheath glia that wrap around these receptive endings. Importantly, ablation of the AFD neuron abrogates glial GFP accumulation, suggesting that glia are taking up neuronal material. We show that this engulfment process is independent of known apoptotic engulfment genes, suggesting that glia employ a novel machinery to regulate uptake of neuronal content. We have previously demonstrated (see abstract by Singhvi and Shaham, this meeting) that AFD-glia interactions are molecularly similar to interactions in the mammalian retina between photoreceptor neurons and the glia-like retinal pigmented epithelium (RPE). Strikingly, outer segments of photoreceptor cells are regularly engulfed by the RPE, suggesting deep parallels between the C. elegans and retinal sensory structures, and also suggesting that C. elegans could be used to understand retinal outer segment pruning. To begin to test this idea, we performed candidate and forward genetic screens that together uncover eleven independent mutants that disrupt glial engulfment of AFD neuron material. We present preliminary characterization and cloning of some of these mutants. Our work suggests that pruning of receptive endings may be an evolutionarily conserved function of sensory glia to regulate the structure and function of sensory receptive endings.

Singhvi, Aakanksha et al. (2015) International Worm Meeting "Glial cues and neuronal cGMP together regulate sensory neuron receptive ending shape and function."

Singhvi, Aakanksha, Shaham, Shai

[International Worm Meeting, 2015]

Neural circuit information processing is initiated when a neuron receives input through its receptive endings from either the environment or other neurons. Receptive endings of central nervous system neurons (e.g. dendritic spines) in mammals are highly malleable and their shapes are regulated by experience. Dynamic morphological properties of sensory neuron receptive endings are much less understood. We have used the microvilli comprising receptive ending of the C. elegans AFD sensory neuron as a model for studying sensory neuron receptive ending shape control. We demonstrate that while neuronal activity does not directly regulate AFD microvilli elongation, glia provide important cues that modulate AFD receptive ending morphology. The glial K-Cl co-transporter KCC-3 and the thrombospondin-domain protein FIG-1 both promote neuronal receptive ending growth. These glial cues require the AFD-expressed receptor guanylyl cyclase GCY-8. GCY-8, along with the phosphodiesterases PDE-1 and PDE-5, in turn regulates receptive ending shape by modulating levels of the second messenger cGMP in AFD. Structure-function studies show that the extracellular domain of GCY-8 plays an important role in inhibiting guanylyl cyclase activity, and biochemical studies bolster our genetic results suggesting that increased cGMP signaling leads to receptive ending regression. Finally, we show that cGMP acts through the actin polymerizing protein WSP-1 to regulate receptive ending elongation. The detailed mechanistic framework we uncover for glial control of receptive ending shape appears to parallel interactions between the glial-like retinal pigmented epithelium and sensory photoreceptor cells in the vertebrate eye. Our work thus reveals that glial regulation of receptive ending shape and function may be a conserved feature of sensory, and perhaps central, receptive endings across species.

Aakanksha Singhvi et al. (2005) International Worm Meeting "A role for tbx-2 in the HSN/PHB neuronal lineage"

Gian Garriga, Aakanksha Singhvi

[International Worm Meeting, 2005]

T-box (Tbx) transcription factors comprise an evolutionarily conserved family of proteins involved in specifying tissue identity during embryonic development. Members of the Tbx2 subfamily function primarily as transcriptional repressors and regulate multiple events during cardiac and limb development in vertebrates. In C.elegans, tbx-2 functions in pharyngeal development, olfactory adaptation and male ray assembly. It is most closely related to the human TBX3 and mouse Tbx2. Haplo-insufficiency of TBX3 leads to ulnar-mammary syndrome, affecting limb, apocrine gland, tooth, heart and genital development. We identified a tbx-2 allele in a screen for mutants with defects in the development of HSN neurons. This allele carries a missense mutation in the T-box DNA binding sequence and behaves similarly to the deletion allele tbx-2(ok529). The HSN neurons often fail to migrate properly and are occasionally missing. The sister cell PHB also appears to be occasionally missing. We find that a mutation in the apoptotic gene ced-3 specifically rescues the missing PHB, suggesting that the PHBs die in the tbx-2 mutants. HAM-1 is a novel protein that regulates the asymmetric division of the neuroblast which generates the HSN/PHB precursor, and ham-1 mutants are also occasionally missing HSN and PHB neurons. While mutations in either ham-1 or ttbx-2 result in the partial loss of HSN and PHB neurons, the double mutants have very few HSN or PHB neurons. These observations indicate that HAM-1 and TBX-2 act in parallel to specify the fates of the HSN and PHB neurons. We are currently asking three questions. First, are the HSN and PHB neurons in the tbx-2; ham-1 double mutants dying? Second, does tbx-2 directly regulate genes of the apoptotic pathway? Third, does TBX-2 function cell-autonomously in the HSN/PHB lineage? If so, does it act in the HSN and PHB neurons or does it function in their progenitors?

Aakanksha Singhvi et al. (2006) Development & Evolution Meeting "Death and Differentiation: Two roles for TBX-2 in the HSN/PHB lineage"

Gian Garriga, Aakanksha Singhvi

[Development & Evolution Meeting, 2006]

Apoptosis is an integral feature of neural development across species. However, little is known about the molecular mechanisms regulating a cell"s ability to “choose” between proliferation, differentiation or apoptosis. We have identified tbx-2 as a gene that regulates aspects of HSN cell migration and survival of its sister neuron, PHB. We propose that at least in the context of the PHB neuron, TBX-2 may be a dual-purpose regulator of both apoptosis and differentiation. TBX-2 and cell death Genetic interactions indicate that tbx-2 acts to inhibit apoptosis in the PHB neuron. Preliminary sequence analysis suggests that the gene encoding the BH3 domain protein EGL-1 has consensus TBX-2 binding sites in its promoter region and thus may be a potential target of TBX-2. We are currently performing experiments to establish whether TBX-2 functions autonomously in the PHB toregulate EGL-1 expression or whether it functions nonautonomously to regulate apoptosis of the PHB indirectly. TBX-2 and cell differentiation HAM-1 was previously identified as a novel asymmetrically distributed protein that indirectly regulates cell fate determination in the HSN/PHB lineage. Our genetic interaction analyses indicate that tbx-2 functions in parallel with ham-1 to regulate differentiation of both the HSN and PHB. We are currently performing experiments to establish whether TBX-2 functions autonomously in the HSN neuron to regulate its migration. TBX-2 orthologs in other species have been implicated in concert with Wnt signaling to regulate cell migrations. We are testing genetically whether the same holds true for HSN migration, which is regulated by Wnts. T-box family transcription factors have so far been shown to regulate either cell fate or senescence in different systems. Their ability to regulate both cell fate and programmed cell death in the same cell, at the same time, suggests a hithero unknown level of complexity in their function and regulation.

Aakanksha Singhvi et al. (2007) International Worm Meeting "Two ARF GTPase cycles and two modes of regulating asymmetric cell divisions."

Gian Garriga, Aakanksha Singhvi, Karla Guitterez, Shaun Cordes

[International Worm Meeting, 2007]

Asymmetric cell divisions in the developing C.elegans nervous system generate both apoptotic cells and differentiated neurons. The mechanisms that specify distinct apoptotic versus neural fates in these dividing cells are largely unknown. We have identified the cytohesin GRP-1, a Guanine Nucleotide Exchange Factor (GEF) for the ARF family of small GTPases; two GTPases, ARF-1 and ARF-6; and the ARF GTPase Activating Protein (GAP) CNT-2 as regulators of the asymmetric division of the Q.p neuroblast. Loss of these molecules results in the survival of Q.pp, a cell normally fated to die, as well as its transformation into the A/PVM-SDQ precursor, its sister cell, resulting in extra A/PVM and SDQ neurons. Homologs of these molecules have been shown to regulate membrane trafficking and cell signaling. Our structure-function analyses show that the GEF activity of GRP-1 is both necessary and sufficient for its function and that the GAP domain of CNT-2 is necessary for its function. Expression of GRP-1 from specific promoters suggests that it functions cell non-autonomously and thus may regulate a signal that controls the Q.p division. GRP-1 localizes to the midbody, raising the interesting possibility that this structure might be involved in cell signaling. Based on several criteria, we come to the surprising conclusion that GRP-1 and CNT-2 define two independent ARF GTPase cycles that regulate asymmetric neuroblast divisions. First, while grp-1 mutations do not alter the sizes of the Q.p daughter cells, cnt-2 mutations do. Second, GRP-1 and CNT-2 have distinct sub-cellular localization patterns. Third, genetic interactions suggest that ARF-1 functions in parallel to GRP-1. Finally, the most compelling evidence indicating that GRP-1 and CNT-2 play different roles in the Q.p division comes from the finding that CNT-2 acts autonomously in the Q.p division, revealing that GRP-1 and CNT-2 act in different cells. Taken together, our studies have for the first time shown in an in vivo context that ARF-mediated pathways regulate asymmetric cell divisions, and that they do so both cell autonomously as well as by regulating a signal.

Ray, Sneha et al. (2021) International Worm Meeting "Specificity in Glia-Neuron Interactions"

Singhvi, Aakanksha, Ray, Sneha

[International Worm Meeting, 2021]

Nervous systems consist of two cell types, neurons and glia, that physically and molecularly interact. One site where glia associate closely with neurons is the neuron receptive endings (NREs), where neurons receive input. Glia can actively regulate NRE shape and function, with consequences on neuron activity and animal behavior. Each glia can associate with multiple NREs. However, whether or how a single glia regulates different associated NREs remains a crucial outstanding question. Unlike most organisms, C. elegans has invariant glia-neuron cell-cell contacts and each cell is born of invariant lineages. This makes it a uniquely tractable system to investigate specificity of individual glia-NRE interactions. To examine this, we focused on the C. elegans amphid sheath (AMsh) glia, which interacts with the NREs of 12 different sensory neurons, including the thermosensory AFD neuron, odor-sensing AWC neuron, and taste-sensing ASE neuron. Ablation of AMsh glia impacts most associated NRE shapes and/or function. We found that AMsh glia localizes the cation chloride cotransporter KCC-3 to a molecular micro-domain only around AFD-NRE, hinting at non-uniform interaction between AMsh glia and different NREs. Indeed, while AMsh glial KCC-3 regulates AFD NRE shape and associated thermosensory behavior, it notably does not affect ASE or AWC NRE shape, or ASE-mediated behavior. To probe how this specificity is achieved, we sought to determine how AMsh KCC-3 localizes to a glial micro-domain. Surprisingly, our genetic and laser ablation studies reveal that the AFD neuron does not recruit KCC-3 localization. Instead, disrupting microtubule-based cilia, present on all amphid NREs, is required to localize glial KCC-3 around AFD-NRE. We are currently investigating the role of individual amphid NREs on KCC-3 localization. Our structure-function studies also identify a 89 amino-acid sequence on KCC-3 that guides its localization. Current studies aim to localize this further and determine interacting partners. These studies show that C. elegans AMsh glia regulate different NREs differently by localizing specific regulatory molecules to single NRE contact sites, and aim to shed light on the underlying molecular mechanism.

Martin, Cecilia et al. (2021) International Worm Meeting "The BAG2 co-chaperone UNC-23 regulates amphid sensory morphology"

Martin, Cecilia, Singhvi, Aakanksha

[International Worm Meeting, 2021]

Cell shape and cell-cell contacts are established during development and then maintained through animal life. More specifically, coordinated multi-tissue regulation is needed for proper organ function, but how this is maintained across cell types is not well understood. To study cell-shape maintenance, we focused on a single neuron in C. elegans, the AFD thermosensory neuron. The neuron receptive ending (NRE) of the AFD is made of many microvilli and a single cilium, both of which are essential for neuron activity. A forward genetic screen for mutants with defective AFD-NRE shape identified a role for UNC-23, the BAG2/Hsp70 co-chaperone. unc-23 mutants have overgrown AFD NRE, a phenotype we term meander. These defects begin as the animal reaches adulthood and are progressive with animal age. The AFD NRE is embedded in the surrounding AMsh glia apical end and we have found similar meander of this apical ending and other glia associated NREs. Intriguingly, cell-specific rescue studies revealed that UNC-23's function is required not in AFD or AMsh, but non-autonomously in the overlying epidermis. Thus, regulation of proteostasis in skin helps maintain sensory glia-neuron shape. Growth of unc-23 mutants in liquid culture and mutations that result in loss of movement both rescue the AFD NRE meander. This suggests that epidermal UNC-23 is dependent on external forces experienced by the worm. Further, unc-23 mutants have expanded epidermal and AMsh apical domains as well as aberrant positioning of junctional markers, AJM-1 and DLG-1. Additionally, the basolateral alpha-integrin, INA-1, is lost from the anterior head region. Finally, mutations in the FGF receptor, EGL-15, or ligand, EGL-17, suppress unc-23 NRE defects. Since integrins regulate apical polarity and FGF signaling, we favor the model where integrin-dependent epidermal polarity and mechanical tension dictates associated glia-neuron shape. Intriguingly, we also found that reduced AFD neuron activity causes meanders and increasing AFD activity rescues the unc-23 meandering defects. Thus, our working model is that skin UNC-23 dictates epidermal apicobasal polarity, causing altered neuron activity and thereby glia-neuron shape. These findings make the exciting implication that coordinated proteostasis maintenance of epidermal polarity regulates associated glia-neuron shape and sensorineural decline with aging

Sun LO et al. (2016) Cell "Glia Get Neurons in Shape."

Barres BA, Sun LO

[Cell, 2016]

Glial cells are essential components of the nervous system. In this issue, Singhvi etal. uncover cellular and molecular mechanisms through which C.elegans glia shape sensory neuron terminals and thus control animal thermosensing behaviors.

Teuliere, Jerome et al. (2011) International Worm Meeting "Arf GTPases antagonize the Frizzled/Planar cell polarity pathway to regulate the asymmetric cell division of the Q.p neuroblast."

Teuliere, Jerome, Talavera, Karla, Singhvi, Aakanksha, Cordes, Shaun, Garriga, Gian

[International Worm Meeting, 2011]

Much of the cellular diversity in the metazoan nervous system is generated by asymmetric cell divisions (ACDs), in which a mother cell divides to produce daughters that adopt different fates. While the regulation of asymmetric divisions of neuroblasts has been well studied in the fruit fly Drosophila melanogaster, little is known about the regulation of these divisions in other organisms. Since all neurons in the nematode Caenorhabditis elegans are produced from asymmetric divisions, the worm is an attractive system to address this question. The Q.p neuroblast normally divides to generate a smaller cell that dies (Q.pp) and a larger precursor (Q.pa) that divides to produce the A/PVM and SDQ neurons. We have identified several genes required for the Q.p division. Mutations in these genes caused the Q.p division to produce two precursors that were more similar in size. Several of these genes encode components of the Arf GTPase cycle, which is involved in membrane trafficking. Loss of the Arf GAP CNT-2 in particular strongly altered the Q.p division, generating two daughters of equivalent size. Analysis of RME-2 in cnt-2 mutant oocytes showed that CNT-2 regulates receptor-mediated endocytosis, and RNAi knockdown of endocytosis genes disrupts the Q.p division, suggesting that CNT-2 mediates endocytic events necessary for the Q.p ACD. What might CNT-2 regulate? We have also implicated the LIN-17 and MOM-5 Frizzled receptors in the Q.p ACD: lin-17 mom-5 double mutants but not lin-17 or mom-5 single mutants displayed Q.p lineage defects, but Wnts were not needed for this regulation. From genetic interactions analysis, cell-autonomy studies and protein localization data, we propose that CNT-2 negatively regulates a Frizzled/Planar Cell Polarity pathway.