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 A et al. (2010) Neuronal Development, Synaptic Function and Behavior, Madison, WI "Glia are required for neuronal receptive ending shape"

Shaham S, Singhvi A

[Neuronal Development, Synaptic Function and Behavior, Madison, WI, 2010]

Formation and plasticity of neural circuits require precise regulation of dendrite and receptive ending shapes. In adult vertebrates, dendrite shapes are remodeled in a controlled manner during sensory deprivation, information processing (synaptic plasticity) or hormonal surges (e.g. ovulation), and perturbations to dendrite morphologies are correlated with cognitive disorders. Similarly, the shapes of sensory receptive endings in C. elegans are altered by sensory deprivation and pheromones. Glia are implicated in the control of neuronal receptive ending shape, however a detailed understand of relevant glia-neuron interactions is lacking. We are using the C. elegans amphid sheath glia and AFD neurons to identify genes underlying glial control of neuronal receptive ending architecture. Previous cell ablation studies from our lab suggest that sheath glia are required for development of the micorvillar-like receptive endings of the AFD thermosensory neuron. We showed that transiently blocking excocytosis in glia by expressing a RAB-1 dominant negative protein promotes loss of AFD fingers, suggesting that an acute signal from glia is required for the maintenance of AFD receptive ending morphology. To identify genes involved in glia-AFD signaling, we conducted a forward genetic screen for mutants with defects in AFD receptive ending morphology. Three independent mutants lacking AFD microvilli have been isolated. Further characterization of these should shed light on the biology of glia, cell shape regulation, and cell-cellcommunication in the nervous system.

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?

Singhvi, A et al. (2017) International Worm Meeting "C. elegans glia regulate neuron shape and function through multiple novel mechanisms including engulfment of neuronal endings."

Shaham, S, Bae, A, Singhvi, A

[International Worm Meeting, 2017]

Mammalian glia prune neuronal synapses, a process that is thought to regulate the number and fidelity of functional neuronal endings and neuron-neuron connections. Glia-mediated pruning is implicated in the control of synapse numbers in normal development, learning and memory, and aging. Disrupted pruning is often seen in neurodevelopmental disorders such as autism, and in age-dependent neurodegenerative disorders such as Alzheimer's disease. Molecular mechanisms regulating pruning are poorly understood. We discovered that C. elegans glia engulf sensory-neuron receptive-ending fragments. Thus, glia-dependent pruning is an evolutionarily conserved phenomenon. Specifically, animals expressing GFP targeted to the receptive-ending of AFD thermosensory neurons also display GFP fluorescence in punctate structures within AMsh glia, which wrap around these receptive-endings. Ablation of the ipsilateral AFD neuron abrogates glial GFP accumulation, suggesting that glia take up material from the ensheathed AFD neuron. We find that glial engulfment rates track with temperature, but not with age, suggesting that glia are responding to AFD activity in a context-dependent manner throughout animal life. CED-1/MEGF10 is required for phagocytic engulfment of apoptotic corpses in C. elegans and for glia-dependent pruning in mammals. Surprisingly, we find that CED-1 is not required for glial engulfment of AFD fragments. However, our analyses implicate the CED-2/5/10/12 ternary complex in glial engulfment, and show that CED-10/Rac GTPase function is required cell-autonomously in glia. Thus, glial engulfment employs components of the apoptotic cell engulfment machinery acting downstream of unidentified receptors. Our preliminary data suggest roles for the phosphatidyly-serine receptor, PSR-1 and FIG-1, a glial thrombospondin-domain protein, in this process. We will also present identification and characterization of additional mutants obtained from a genetic screen for disruption of glia-dependent engulfment. In a previous study (Singhvi et al, Cell, 2016), we identified a novel mechanism whereby glia regulate the shape and function of neuronal receptive-endings by regulating the ionic microenvironment, and described the underlying molecular pathway. We also showed that glia molecularly discriminate between associated neurons to differentially regulate each one. Together, our studies reveal that a single glial cell modulates the shape and function of its associated neurons through multiple cellular and molecular mechanisms.

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.

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.

Teuliere, Jerome et al. (2013) International Worm Meeting "Several ArfGEFs regulate the apoptotic fate in Q neuroblast asymmetric cell divisions."

Cordes, Shaun, Garriga, Gian, Teuliere, Jerome

[International Worm Meeting, 2013]

Arf guanine nucleotide exchange factors (GEFs) can regulate cell adhesion, actin cytoskeletal dynamics, and membrane trafficking through the activation of the Arf small GTPases. We previously found that an Arf GTPase Activating Protein (GAP), CNT-2, was essential in controlling cell size and the cell death fate in the Q neuroblast lineage (Singhvi et al, 2011). We report here that several ArfGEFs control the asymmetric divisions of the Q cell daughters. Q.a and Q.p are both neuroblasts that divide to produce a larger neuronal precursor or neuron and a smaller cell fated to die. Loss of the cytohesin ArfGEF homolog GRP-1 resulted in the production of daughter cells that are more similar in size and in the transformation of the apoptotic daughter into its sister, resulting in the production of extra neurons. GRP-1's GEF activity, mediated by its SEC7 domain, is necessary for its role in the Q.p neuroblast. The loss of GRP-1, however, resulted in a phenotype that was less severe than the loss of CNT-2, suggesting that additional ArfGEFs remained to be identified. By screening for grp-1 enhancers, we indeed identified two more ArfGEFs, EFA-6 and M02B7.5/Schizo/IQSEC, that are necessary for the Q.p division. Functional GFP-tagged GRP-1 proteins localized primarily to the nucleus, but targeting the GRP-1 SEC7 domain to different cellular compartments suggests that GRP-1 acts at the cell cortex. EFA-6 localized at the cell cortex, suggesting a functions there as well. Surprisingly, loss of GRP-1 and CNT-2 resulted in identical Q.a phenotypes, and neither efa-6 nor M02B7.5 appears to function in this division. We will propose models that explain the differential involvement of these ArfGEFs in these two asymmetric divisions. Singhvi et al., Curr Biol. 2011 Jun 7;21(11):948-54.

Martin, C. et al. (2019) International Worm Meeting "The BAG2 co-chaperone UNC-23 regulates amphid sensory neuron morphology."

Singhvi, A., Martin, C.

[International Worm Meeting, 2019]

Environmental stimuli are received by specialized receptive endings (NREs) on sensory neurons, which house the molecular machinery required for sensation. Information is then transmitted to, and processed via neural circuits to result in behavior. The function of sensory NREs is dependent on proper development and maintenance of their morphology. To study dynamic NRE morphology, we are using the C. elegans AFD thermosensory NRE as a model. The AFD-NRE is a complex microvilli structure, and is ensheathed by the AMsh glia cell. From a forward genetic screen, we found that UNC-23, an HSP-70 co-chaperone is required for proper AFD-NRE morphology. In unc-23 adult mutants animals, AFD-NRE microvilli exhibit an aberrant and progressive increase in length. Other sensory NREs associated with AMsh glia also show similar defects. Interestingly, cell-specific rescues of unc-23 in AFD neuron and AMsh glia suggest that UNC-23 [SA1] is required in glia but not in neurons for function. Consistent with this, we observe abnormally large vesicular structures within glia, suggestive of a secretory block. In addition, we found that a mutation in chn-1, an E3/E4 ubiquitin ligase that binds HSP-70 to degrade unfolded polypeptide, partially suppresses AFD-NRE defects. Mutations in chn-1 also show defects in AFD-NRE mediated animal behavior. Taken together, we hypothesize that UNC-23/ CHN-1 mediated regulation of protein folding in AMsh glia modulates AFD-NRE microvilli length, presumably by regulating the turnover of an unknown polypeptide. We are now testing this model by performing molecular genetic and biochemical experiments to identify UNC-23 functions and targets in glia. In conclusion, this study shows that regulation of proteostatic function in AMsh glia is essential for maintenance of associated sensory NRE morphology through animal life.