Publications by authors named "Shai Shaham"

76 Publications

Disabling the Fanconi Anemia Pathway in Stem Cells Leads to Radioresistance and Genomic Instability.

Cancer Res 2021 May 3. Epub 2021 May 3.

Laboratory of Signal Transduction, Memorial Sloan Kettering Cancer Center

Fanconi Anemia (FA) is an inherited genome instability syndrome characterized by interstrand crosslink hypersensitivity, congenital defects, bone marrow failure, and cancer predisposition. Although DNA repair mediated by FA genes has been extensively studied, how inactivation of these genes leads to specific cellular phenotypic consequences associated with FA is not well understood. Here we report that FA stem cells in the C. elegans germline and in murine embryos display marked nonhomologous end joining (NHEJ)-dependent radiation resistance, leading to survival of progeny cells carrying genetic lesions. By contrast, DNA crosslinking does not induce generational genomic instability in FA stem cells, as widely accepted, but rather drives NHEJ-dependent apoptosis in both species. These findings suggest that FA is a stem cell disease reflecting inappropriate NHEJ, which is mutagenic and carcinogenic as a result of DNA misrepair, while marrow failure represents hematopoietic stem cell apoptosis.
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http://dx.doi.org/10.1158/0008-5472.CAN-20-3309DOI Listing
May 2021

Glia actively sculpt sensory neurons by controlled phagocytosis to tune animal behavior.

Elife 2021 03 24;10. Epub 2021 Mar 24.

Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, United States.

Glia in the central nervous system engulf neuron fragments to remodel synapses and recycle photoreceptor outer segments. Whether glia passively clear shed neuronal debris or actively prune neuron fragments is unknown. How pruning of single-neuron endings impacts animal behavior is also unclear. Here, we report our discovery of glia-directed neuron pruning in Adult AMsh glia engulf sensory endings of the AFD thermosensory neuron by repurposing components of the conserved apoptotic corpse phagocytosis machinery. The phosphatidylserine (PS) flippase TAT-1/ATP8A functions with glial PS-receptor PSR-1/PSR and PAT-2/α-integrin to initiate engulfment. This activates glial CED-10/Rac1 GTPase through the ternary GEF complex of CED-2/CrkII, CED-5/DOCK180, CED-12/ELMO. Execution of phagocytosis uses the actin-remodeler WSP-1/nWASp. This process dynamically tracks AFD activity and is regulated by temperature, the AFD sensory input. Importantly, glial CED-10 levels regulate engulfment rates downstream of neuron activity, and engulfment-defective mutants exhibit altered AFD-ending shape and thermosensory behavior. Our findings reveal a molecular pathway underlying glia-dependent engulfment in a peripheral sense-organ and demonstrate that glia actively engulf neuron fragments, with profound consequences on neuron shape and animal sensory behavior.
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http://dx.doi.org/10.7554/eLife.63532DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8079151PMC
March 2021

Stress-Induced Neural Plasticity Mediated by Glial GPCR REMO-1 Promotes C. elegans Adaptive Behavior.

Cell Rep 2021 01;34(2):108607

Laboratory of Developmental Genetics, The Rockefeller University, 1230 York Avenue, New York, NY 10065, USA. Electronic address:

Animal nervous systems remodel following stress. Although global stress-dependent changes are well documented, contributions of individual neuron remodeling events to animal behavior modification are challenging to study. In response to environmental insults, C. elegans become stress-resistant dauers. Dauer entry induces amphid sensory organ remodeling in which bilateral AMsh glial cells expand and fuse, allowing embedded AWC chemosensory neurons to extend sensory receptive endings. We show that amphid remodeling correlates with accelerated dauer exit upon exposure to favorable conditions and identify a G protein-coupled receptor, REMO-1, driving AMsh glia fusion, AWC neuron remodeling, and dauer exit. REMO-1 is expressed in and localizes to AMsh glia tips, is dispensable for other remodeling events, and promotes stress-induced expression of the remodeling receptor tyrosine kinase VER-1. Our results demonstrate how single-neuron structural changes affect animal behavior, identify key glial roles in stress-induced nervous system plasticity, and demonstrate that remodeling primes animals to respond to favorable conditions.
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http://dx.doi.org/10.1016/j.celrep.2020.108607DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7845533PMC
January 2021

Behaviorally consequential astrocytic regulation of neural circuits.

Neuron 2021 02 31;109(4):576-596. Epub 2020 Dec 31.

Department of Physiology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095-1751, USA; Department of Neurobiology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095-1751, USA. Electronic address:

Astrocytes are a large and diverse population of morphologically complex cells that exist throughout nervous systems of multiple species. Progress over the last two decades has shown that astrocytes mediate developmental, physiological, and pathological processes. However, a long-standing open question is how astrocytes regulate neural circuits in ways that are behaviorally consequential. In this regard, we summarize recent studies using Caenorhabditis elegans, Drosophila melanogaster, Danio rerio, and Mus musculus. The data reveal diverse astrocyte mechanisms operating in seconds or much longer timescales within neural circuits and shaping multiple behavioral outputs. We also refer to human diseases that have a known primary astrocytic basis. We suggest that including astrocytes in mechanistic, theoretical, and computational studies of neural circuits provides new perspectives to understand behavior, its regulation, and its disease-related manifestations.
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http://dx.doi.org/10.1016/j.neuron.2020.12.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7897322PMC
February 2021

Cell death in animal development.

Development 2020 07 24;147(14). Epub 2020 Jul 24.

Laboratory of Developmental Genetics, The Rockefeller University, 1230 York Avenue, New York, NY 10065, USA

Cell death is an important facet of animal development. In some developing tissues, death is the ultimate fate of over 80% of generated cells. Although recent studies have delineated a bewildering number of cell death mechanisms, most have only been observed in pathological contexts, and only a small number drive normal development. This Primer outlines the important roles, different types and molecular players regulating developmental cell death, and discusses recent findings with which the field currently grapples. We also clarify terminology, to distinguish between developmental cell death mechanisms, for which there is evidence for evolutionary selection, and cell death that follows genetic, chemical or physical injury. Finally, we suggest how advances in understanding developmental cell death may provide insights into the molecular basis of developmental abnormalities and pathological cell death in disease.
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http://dx.doi.org/10.1242/dev.191882DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7390631PMC
July 2020

Development or Disease: Caspases Balance Growth and Immunity in C. elegans.

Dev Cell 2020 05;53(3):259-260

Laboratory of Developmental Genetics, The Rockefeller University, 1230 York Avenue, New York, NY 10065, USA. Electronic address:

Caspase proteases execute apoptosis but also function in development. In this issue of Developmental Cell, Weaver et al. report that C. elegans CED-3 caspase promotes animal growth through PMK-1/p38 kinase cleavage, and at the expense of pathogen and stress immunity, revealing an unexpected homeostatic relationship between development and disease.
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http://dx.doi.org/10.1016/j.devcel.2020.04.006DOI Listing
May 2020

Age-dependent changes in response property and morphology of a thermosensory neuron and thermotaxis behavior in Caenorhabditis elegans.

Aging Cell 2020 05 19;19(5):e13146. Epub 2020 Apr 19.

Institute of Molecular Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan.

Age-dependent cognitive and behavioral deterioration may arise from defects in different components of the nervous system, including those of neurons, synapses, glial cells, or a combination of them. We find that AFD, the primary thermosensory neuron of Caenorhabditis elegans, in aged animals is characterized by loss of sensory ending integrity, including reduced actin-based microvilli abundance and aggregation of thermosensory guanylyl cyclases. At the functional level, AFD neurons in aged animals are hypersensitive to high temperatures and show sustained sensory-evoked calcium dynamics, resulting in a prolonged operating range. At the behavioral level, senescent animals display cryophilic behaviors that remain plastic to acute temperature changes. Excessive cyclase activity of the AFD-specific guanylyl cyclase, GCY-8, is associated with developmental defects in AFD sensory ending and cryophilic behavior. Surprisingly, loss of the GCY-8 cyclase domain reduces these age-dependent morphological and behavioral changes, while a prolonged AFD operating range still exists in gcy-8 animals. The lack of apparent correlation between age-dependent changes in the morphology or stimuli-evoked response properties of primary sensory neurons and those in related behaviors highlights the importance of quantitative analyses of aging features when interpreting age-related changes at structural and functional levels. Our work identifies aging hallmarks in AFD receptive ending, temperature-evoked AFD responses, and experience-based thermotaxis behavior, which serve as a foundation to further elucidate the neural basis of cognitive aging.
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http://dx.doi.org/10.1111/acel.13146DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7253067PMC
May 2020

Learning and Memory: Mind over Matter in C. elegans.

Curr Biol 2019 05;29(10):R365-R367

The Rockefeller University, 1230 York Avenue, New York, NY 10021, USA. Electronic address:

The capacity to respond to adverse conditions is key for animal survival. Research in the nematode Caenorhabditis elegans demonstrates that retrieval of aversive memories, stored within sensory neurons, is sufficient to induce a protective systemic stress response that improves fitness.
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http://dx.doi.org/10.1016/j.cub.2019.04.036DOI Listing
May 2019

Glutamate spillover in C. elegans triggers repetitive behavior through presynaptic activation of MGL-2/mGluR5.

Nat Commun 2019 04 23;10(1):1882. Epub 2019 Apr 23.

Laboratory of Developmental Genetics, The Rockefeller University, 1230 York Avenue, New York, NY, 10065, USA.

Glutamate is a major excitatory neurotransmitter, and impaired glutamate clearance following synaptic release promotes spillover, inducing extra-synaptic signaling. The effects of glutamate spillover on animal behavior and its neural correlates are poorly understood. We developed a glutamate spillover model in Caenorhabditis elegans by inactivating the conserved glial glutamate transporter GLT-1. GLT-1 loss drives aberrant repetitive locomotory reversal behavior through uncontrolled oscillatory release of glutamate onto AVA, a major interneuron governing reversals. Repetitive glutamate release and reversal behavior require the glutamate receptor MGL-2/mGluR5, expressed in RIM and other interneurons presynaptic to AVA. mgl-2 loss blocks oscillations and repetitive behavior; while RIM activation is sufficient to induce repetitive reversals in glt-1 mutants. Repetitive AVA firing and reversals require EGL-30/Gαq, an mGluR5 effector. Our studies reveal that cyclic autocrine presynaptic activation drives repetitive reversals following glutamate spillover. That mammalian GLT1 and mGluR5 are implicated in pathological motor repetition suggests a common mechanism controlling repetitive behaviors.
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http://dx.doi.org/10.1038/s41467-019-09581-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6478929PMC
April 2019

Glia-Neuron Interactions in .

Annu Rev Neurosci 2019 07 18;42:149-168. Epub 2019 Mar 18.

Laboratory of Developmental Genetics, The Rockefeller University, New York, NY 10065, USA; email:

Glia are abundant components of animal nervous systems. Recognized 170 years ago, concerted attempts to understand these cells began only recently. From these investigations glia, once considered passive filler material in the brain, have emerged as active players in neuron development and activity. Glia are essential for nervous system function, and their disruption leads to disease. The nematode possesses glial types similar to vertebrate glia, based on molecular, morphological, and functional criteria, and has become a powerful model in which to study glia and their neuronal interactions. Facile genetic and transgenic methods in this animal allow the discovery of genes required for glial functions, and effects of glia at single synapses can be monitored by tracking neuron shape, physiology, or animal behavior. Here, we review recent progress in understanding glia-neuron interactions in . We highlight similarities with glia in other animals, and suggest conserved emerging principles of glial function.
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http://dx.doi.org/10.1146/annurev-neuro-070918-050314DOI Listing
July 2019

RAB-35 and ARF-6 GTPases Mediate Engulfment and Clearance Following Linker Cell-Type Death.

Dev Cell 2018 10 13;47(2):222-238.e6. Epub 2018 Sep 13.

Laboratory of Developmental Genetics, The Rockefeller University, New York, NY 10065, USA. Electronic address:

Clearance of dying cells is essential for development and homeostasis. Conserved genes mediate apoptotic cell removal, but whether these genes control non-apoptotic cell removal is a major open question. Linker cell-type death (LCD) is a prevalent non-apoptotic developmental cell death process with features conserved from C. elegans to vertebrates. Using microfluidics-based long-term in vivo imaging, we show that unlike apoptotic cells, the C. elegans linker cell, which dies by LCD, is competitively phagocytosed by two neighboring cells, resulting in cell splitting. Subsequent cell elimination does not require apoptotic engulfment genes. Rather, we find that RAB-35 GTPase is a key coordinator of competitive phagocytosis onset and cell degradation. RAB-35 binds CNT-1, an ARF-6 GTPase activating protein, and removes ARF-6, a degradation inhibitor, from phagosome membranes. This facilitates phosphatidylinositol-4,5-bisphosphate removal from phagosome membranes, promoting phagolysosome maturation. Our studies suggest that RAB-35 and ARF-6 drive a conserved program eliminating cells dying by LCD.
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http://dx.doi.org/10.1016/j.devcel.2018.08.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6200590PMC
October 2018

Automated C. elegans embryo alignments reveal brain neuropil position invariance despite lax cell body placement.

PLoS One 2018 28;13(3):e0194861. Epub 2018 Mar 28.

Laboratory of Developmental Genetics, The Rockefeller University, New York, NY, United States of America.

The Caenorhabditis elegans cell lineage is nearly invariant. Whether this stereotyped cell-division pattern promotes reproducibility in cell shapes/positions is not generally known, as manual spatiotemporal cell-shape/position alignments are labor-intensive, and fully-automated methods are not described. Here, we report automated algorithms for spatiotemporal alignments of C. elegans embryos from pre-morphogenesis to motor-activity initiation. We use sparsely-labeled green-fluorescent nuclei and a pan-nuclear red-fluorescent reporter to register consecutive imaging time points and compare embryos. Using our method, we monitor early assembly of the nerve-ring (NR) brain neuropil. While NR pioneer neurons exhibit reproducible growth kinetics and axon positions, cell-body placements are variable. Thus, pioneer-neuron axon locations, but not cell-body positions, are under selection. We also show that anterior NR displacement in cam-1/ROR Wnt-receptor mutants is not an early NR assembly defect. Our results demonstrate the utility of automated spatiotemporal alignments of C. elegans embryos, and uncover key principles guiding nervous-system development in this animal.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0194861PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5874040PMC
July 2018

EFF-1 fusogen promotes phagosome sealing during cell process clearance in Caenorhabditis elegans.

Nat Cell Biol 2018 04 19;20(4):393-399. Epub 2018 Mar 19.

Laboratory of Developmental Genetics, The Rockefeller University, New York, NY, USA.

Phagocytosis of dying cells is critical in development and immunity. Although proteins for recognition and engulfment of cellular debris following cell death are known, proteins that directly mediate phagosome sealing are uncharacterized. Furthermore, whether all phagocytic targets are cleared using the same machinery is unclear. Degeneration of morphologically complex cells, such as neurons, glia and melanocytes, produces phagocytic targets of various shapes and sizes located in different microenvironments. Thus, such cells offer unique settings to explore engulfment programme mechanisms and specificity. Here, we report that dismantling and clearance of a morphologically complex Caenorhabditis elegans epithelial cell requires separate cell soma, proximal and distal process programmes. Similar compartment-specific events govern the elimination of a C. elegans neuron. Although canonical engulfment proteins drive cell soma clearance, these are not required for process removal. We find that EFF-1, a protein previously implicated in cell-cell fusion , specifically promotes distal process phagocytosis. EFF-1 localizes to phagocyte pseudopod tips and acts exoplasmically to drive phagosome sealing. eff-1 mutations result in phagocytosis arrest with unsealed phagosomes. Our studies suggest universal mechanisms for dismantling morphologically complex cells and uncover a phagosome-sealing component that promotes cell process clearance.
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http://dx.doi.org/10.1038/s41556-018-0068-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5876135PMC
April 2018

Glia Modulate a Neuronal Circuit for Locomotion Suppression during Sleep in C. elegans.

Cell Rep 2018 03;22(10):2575-2583

Laboratory of Developmental Genetics, The Rockefeller University, 1230 York Avenue, New York, NY 10065, USA. Electronic address:

Glia have been suggested to regulate sleep-like states in vertebrates and invertebrates alike. In the nematode Caenorhabditis elegans, sleep is associated with molting between larval stages. To understand if glia modulate neural circuits driving sleep in C. elegans larvae, we ablated the astrocyte-like CEPsh glia. We found that glia-ablated animals exhibit episodes of immobility preceding sleep, prolonged sleep, molting-independent short-duration locomotory pausing, and delayed development. CEPsh glia ensheath synapses between the sleep-associated ALA neuron and its postsynaptic partner AVE, a major locomotion interneuron. While AVE calcium transients normally correlate with head retraction, glia ablation results in prolonged calcium transients that are uncoupled from movement. Strikingly, all these glia ablation defects are suppressed by the ablation of ALA. Our results suggest that glia attenuate sleep-promoting inhibitory connections between ALA and AVE, uncovering specific roles for glia in sleep behavior. We propose that similar mechanisms may underlie glial roles in sleep in other animals.
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http://dx.doi.org/10.1016/j.celrep.2018.02.036DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5870883PMC
March 2018

Glia initiate brain assembly through noncanonical Chimaerin-Furin axon guidance in C. elegans.

Nat Neurosci 2017 Oct 28;20(10):1350-1360. Epub 2017 Aug 28.

Laboratory of Developmental Genetics, The Rockefeller University, New York, New York, USA.

Brain assembly is hypothesized to begin when pioneer axons extend over non-neuronal cells, forming tracts guiding follower axons. Yet pioneer-neuron identities, their guidance substrates, and their interactions are not well understood. Here, using time-lapse embryonic imaging, genetics, protein-interaction, and functional studies, we uncover the early events of C. elegans brain assembly. We demonstrate that C. elegans glia are key for assembly initiation, guiding pioneer and follower axons using distinct signals. Pioneer sublateral neurons, with unique growth properties, anatomy, and innervation, cooperate with glia to mediate follower-axon guidance. We further identify a Chimaerin (CHIN-1)- Furin (KPC-1) double-mutant that severely disrupts assembly. CHIN-1 and KPC-1 function noncanonically, in glia and pioneer neurons, for guidance-cue trafficking. We exploit this bottleneck to define roles for glial Netrin and Semaphorin in pioneer- and follower-axon guidance, respectively, and for glial and pioneer-neuron Flamingo (CELSR) in follower-axon navigation. Taken together, our studies reveal previously undescribed glial roles in pioneer-axon guidance, suggesting conserved principles of brain assembly.
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http://dx.doi.org/10.1038/nn.4630DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5614858PMC
October 2017

IGDB-2, an Ig/FNIII protein, binds the ion channel LGC-34 and controls sensory compartment morphogenesis in C. elegans.

Dev Biol 2017 10 10;430(1):105-112. Epub 2017 Aug 10.

Laboratory of Developmental Genetics, The Rockefeller University, 1230 York Avenue, New York, NY 10065, USA. Electronic address:

Sensory organ glia surround neuronal receptive endings (NREs), forming a specialized compartment important for neuronal activity, and reminiscent of glia-ensheathed synapses in the central nervous system. We previously showed that DAF-6, a Patched-related protein, is required in glia of the C. elegans amphid sensory organ to restrict sensory compartment size. LIT-1, a Nemo-like kinase, and SNX-1, a retromer component, antagonize DAF-6 and promote compartment expansion. To further explore the machinery underlying compartment size control, we sought genes whose inactivation restores normal compartment size to daf-6 mutants. We found that mutations in igdb-2, encoding a single-pass transmembrane protein containing Ig-like and fibronectin type III domains, suppress daf-6 mutant defects. IGDB-2 acts in glia, where it localizes to glial membranes surrounding NREs, and, together with LIT-1 and SNX-1, regulates compartment morphogenesis. Immunoprecipitation followed by mass spectrometry demonstrates that IGDB-2 binds to LGC-34, a predicted ligand-gated ion channel, and lgc-34 mutations inhibit igdb-2 suppression of daf-6. Our findings reveal a novel membrane protein complex and suggest possible mechanisms for how sensory compartment size is controlled.
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http://dx.doi.org/10.1016/j.ydbio.2017.08.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5593787PMC
October 2017

Sensory Cilia: Generating Diverse Shapes One Ig Domain at a Time.

Curr Biol 2017 07;27(13):R654-R656

The Rockefeller University, 1230 York Avenue, New York, NY 10021, USA. Electronic address:

How morphologically complex cilia form is not well understood. A key regulator of ciliary shape has now been identified that links the establishment of neuronal fate with the formation of cell-specific ciliary structures in Caenorhabditis elegans.
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http://dx.doi.org/10.1016/j.cub.2017.05.051DOI Listing
July 2017

Non-apoptotic cell death in animal development.

Cell Death Differ 2017 08 17;24(8):1326-1336. Epub 2017 Feb 17.

Laboratory of Developmental Genetics, The Rockefeller University, 1230 York Avenue, New York, NY 10065, USA.

Programmed cell death (PCD) is an important process in the development of multicellular organisms. Apoptosis, a form of PCD characterized morphologically by chromatin condensation, membrane blebbing, and cytoplasm compaction, and molecularly by the activation of caspase proteases, has been extensively investigated. Studies in Caenorhabditis elegans, Drosophila, mice, and the developing chick have revealed, however, that developmental PCD also occurs through other mechanisms, morphologically and molecularly distinct from apoptosis. Some non-apoptotic PCD pathways, including those regulating germ cell death in Drosophila, still appear to employ caspases. However, another prominent cell death program, linker cell-type death (LCD), is morphologically conserved, and independent of the key genes that drive apoptosis, functioning, at least in part, through the ubiquitin proteasome system. These non-apoptotic processes may serve as backup programs when caspases are inactivated or unavailable, or, more likely, as freestanding cell culling programs. Non-apoptotic PCD has been documented extensively in the developing nervous system, and during the formation of germline and somatic gonadal structures, suggesting that preservation of these mechanisms is likely under strong selective pressure. Here, we discuss our current understanding of non-apoptotic PCD in animal development, and explore possible roles for LCD and other non-apoptotic developmental pathways in vertebrates. We raise the possibility that during vertebrate development, apoptosis may not be the major PCD mechanism.
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http://dx.doi.org/10.1038/cdd.2017.20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5520451PMC
August 2017

Infrared laser-induced gene expression for tracking development and function of single C. elegans embryonic neurons.

Nat Commun 2017 01 18;8:14100. Epub 2017 Jan 18.

Laboratory of Developmental Genetics, The Rockefeller University, 1230 York Avenue, New York, New York 10065, USA.

Visualizing neural-circuit assembly in vivo requires tracking growth of optically resolvable neurites. The Caenorhabditis elegans embryonic nervous system, comprising 222 neurons and 56 glia, is attractive for comprehensive studies of development; however, embryonic reporters are broadly expressed, making single-neurite tracking/manipulation challenging. We present a method, using an infrared laser, for reproducible heat-dependent gene expression in small sublineages (one to four cells) without radiation damage. We go beyond proof-of-principle, and use our system to label and track single neurons during early nervous-system assembly. We uncover a retrograde extension mechanism for axon growth, and reveal the aetiology of axon-guidance defects in sax-3/Robo and vab-1/EphR mutants. We also perform cell-specific rescues, determining DAF-6/patched-related site of action during sensory-organ development. Simultaneous ablation and labelling of cells using our system reveals roles for glia in dendrite extension. Our method can be applied to other optically/IR-transparent organisms, and opens the door to high-resolution systematic analyses of C. elegans morphogenesis.
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http://dx.doi.org/10.1038/ncomms14100DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5253673PMC
January 2017

Long-Term High-Resolution Imaging of Developing C. elegans Larvae with Microfluidics.

Dev Cell 2017 01 29;40(2):202-214. Epub 2016 Dec 29.

Center for Physics and Biology, The Rockefeller University, New York, NY 10065, USA. Electronic address:

Long-term studies of Caenorhabditis elegans larval development traditionally require tedious manual observations because larvae must move to develop, and existing immobilization techniques either perturb development or are unsuited for young larvae. Here, we present a simple microfluidic device to simultaneously follow development of ten C. elegans larvae at high spatiotemporal resolution from hatching to adulthood (∼3 days). Animals grown in microchambers are periodically immobilized by compression to allow high-quality imaging of even weak fluorescence signals. Using the device, we obtain cell-cycle statistics for C. elegans vulval development, a paradigm for organogenesis. We combine Nomarski and multichannel fluorescence microscopy to study processes such as cell-fate specification, cell death, and transdifferentiation throughout post-embryonic development. Finally, we generate time-lapse movies of complex neural arborization through automated image registration. Our technique opens the door to quantitative analysis of time-dependent phenomena governing cellular behavior during C. elegans larval development.
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http://dx.doi.org/10.1016/j.devcel.2016.11.022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5263027PMC
January 2017

A High-Throughput Small Molecule Screen for C. elegans Linker Cell Death Inhibitors.

PLoS One 2016 7;11(10):e0164595. Epub 2016 Oct 7.

Laboratory of Developmental Genetics, The Rockefeller University, New York, New York, United States of America.

Programmed cell death is a ubiquitous process in metazoan development. Apoptosis, one cell death form, has been studied extensively. However, mutations inactivating key mammalian apoptosis regulators do not block most developmental cell culling, suggesting that other cell death pathways are likely important. Recent work in the nematode Caenorhabditis elegans identified a non-apoptotic cell death form mediating the demise of the male-specific linker cell. This cell death process (LCD, linker cell-type death) is morphologically conserved, and its molecular effectors also mediate axon degeneration in mammals and Drosophila. To develop reagents to manipulate LCD, we established a simple high-throughput screening protocol for interrogating the effects of small molecules on C. elegans linker cell death in vivo. From 23,797 compounds assayed, 11 reproducibly block linker cell death onset. Of these, five induce animal lethality, and six promote a reversible developmental delay. These results provide proof-of principle validation of our screening protocol, demonstrate that developmental progression is required for linker cell death, and suggest that larger scale screens may identify LCD-specific small-molecule regulators that target the LCD execution machinery.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0164595PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5055323PMC
June 2017

A secreted bacterial peptidoglycan hydrolase enhances tolerance to enteric pathogens.

Science 2016 09 22;353(6306):1434-1437. Epub 2016 Sep 22.

Laboratory of Chemical Biology and Microbial Pathogenesis, The Rockefeller University, New York, NY 10065, USA.

The intestinal microbiome modulates host susceptibility to enteric pathogens, but the specific protective factors and mechanisms of individual bacterial species are not fully characterized. We show that secreted antigen A (SagA) from Enterococcus faecium is sufficient to protect Caenorhabditis elegans against Salmonella pathogenesis by promoting pathogen tolerance. The NlpC/p60 peptidoglycan hydrolase activity of SagA is required and generates muramyl-peptide fragments that are sufficient to protect C. elegans against Salmonella pathogenesis in a tol-1-dependent manner. SagA can also be heterologously expressed and secreted to improve the protective activity of probiotics against Salmonella pathogenesis in C. elegans and mice. Our study highlights how protective intestinal bacteria can modify microbial-associated molecular patterns to enhance pathogen tolerance.
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http://dx.doi.org/10.1126/science.aaf3552DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5158264PMC
September 2016

Transcriptional control of non-apoptotic developmental cell death in C. elegans.

Cell Death Differ 2016 12 29;23(12):1985-1994. Epub 2016 Jul 29.

Laboratory of Developmental Genetics, The Rockefeller University, New York, NY, USA.

Programmed cell death is an essential aspect of animal development. Mutations in vertebrate genes that mediate apoptosis only mildly perturb development, suggesting that other cell death modes likely have important roles. Linker cell-type death (LCD) is a morphologically conserved cell death form operating during the development of Caenorhabditis elegans and vertebrates. We recently described a molecular network governing LCD in C. elegans, delineating a key role for the transcription factor heat-shock factor 1 (HSF-1). Although HSF-1 functions to protect cells from stress in many settings by inducing expression of protein folding chaperones, it promotes LCD by inducing expression of the conserved E2 ubiquitin-conjugating enzyme LET-70/UBE2D2, which is not induced by stress. Following whole-genome RNA interference and candidate gene screens, we identified and characterized four conserved regulators required for LCD. Here we show that two of these, NOB-1/Hox and EOR-1/PLZF, act upstream of HSF-1, in the context of Wnt signaling. A third protein, NHR-67/TLX/NR2E1, also functions upstream of HSF-1, and has a separate activity that prevents precocious expression of HSF-1 transcriptional targets. We demonstrate that the SET-16/mixed lineage leukemia 3/4 (MLL3/4) chromatin regulation complex functions at the same step or downstream of HSF-1 to control LET-70/UBE2D2 expression. Our results identify conserved proteins governing LCD, and demonstrate that transcriptional regulators influence this process at multiple levels.
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http://dx.doi.org/10.1038/cdd.2016.77DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5136488PMC
December 2016

Maintenance and propagation of a deleterious mitochondrial genome by the mitochondrial unfolded protein response.

Nature 2016 05 2;533(7603):416-9. Epub 2016 May 2.

Cell Biology Program, Memorial Sloan Kettering Cancer Center, New York, New York 10065, USA.

Mitochondrial genomes (mitochondrial DNA, mtDNA) encode essential oxidative phosphorylation (OXPHOS) components. Because hundreds of mtDNAs exist per cell, a deletion in a single mtDNA has little impact. However, if the deletion genome is enriched, OXPHOS declines, resulting in cellular dysfunction. For example, Kearns-Sayre syndrome is caused by a single heteroplasmic mtDNA deletion. More broadly, mtDNA deletion accumulation has been observed in individual muscle cells and dopaminergic neurons during ageing. It is unclear how mtDNA deletions are tolerated or how they are propagated in somatic cells. One mechanism by which cells respond to OXPHOS dysfunction is by activating the mitochondrial unfolded protein response (UPR(mt)), a transcriptional response mediated by the transcription factor ATFS-1 that promotes the recovery and regeneration of defective mitochondria. Here we investigate the role of ATFS-1 in the maintenance and propagation of a deleterious mtDNA in a heteroplasmic Caenorhabditis elegans strain that stably expresses wild-type mtDNA and mtDNA with a 3.1-kilobase deletion (∆mtDNA) lacking four essential genes. The heteroplasmic strain, which has 60% ∆mtDNA, displays modest mitochondrial dysfunction and constitutive UPR(mt) activation. ATFS-1 impairment reduced the ∆mtDNA nearly tenfold, decreasing the total percentage to 7%. We propose that in the context of mtDNA heteroplasmy, UPR(mt) activation caused by OXPHOS defects propagates or maintains the deleterious mtDNA in an attempt to recover OXPHOS activity by promoting mitochondrial biogenesis and dynamics.
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http://dx.doi.org/10.1038/nature17989DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4873342PMC
May 2016

PROS-1/Prospero Is a Major Regulator of the Glia-Specific Secretome Controlling Sensory-Neuron Shape and Function in C. elegans.

Cell Rep 2016 Apr 7;15(3):550-562. Epub 2016 Apr 7.

Laboratory of Developmental Genetics, The Rockefeller University, 1230 York Avenue, New York, NY 10065, USA. Electronic address:

Sensory neurons are an animal's gateway to the world, and their receptive endings, the sites of sensory signal transduction, are often associated with glia. Although glia are known to promote sensory-neuron functions, the molecular bases of these interactions are poorly explored. Here, we describe a post-developmental glial role for the PROS-1/Prospero/PROX1 homeodomain protein in sensory-neuron function in C. elegans. Using glia expression profiling, we demonstrate that, unlike previously characterized cell fate roles, PROS-1 functions post-embryonically to control sense-organ glia-specific secretome expression. PROS-1 functions cell autonomously to regulate glial secretion and membrane structure, and non-cell autonomously to control the shape and function of the receptive endings of sensory neurons. Known glial genes controlling sensory-neuron function are PROS-1 targets, and we identify additional PROS-1-dependent genes required for neuron attributes. Drosophila Prospero and vertebrate PROX1 are expressed in post-mitotic sense-organ glia and astrocytes, suggesting conserved roles for this class of transcription factors.
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http://dx.doi.org/10.1016/j.celrep.2016.03.051DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4838487PMC
April 2016

A Glial K/Cl Transporter Controls Neuronal Receptive Ending Shape by Chloride Inhibition of an rGC.

Cell 2016 May 7;165(4):936-48. Epub 2016 Apr 7.

Laboratory of Developmental Genetics, The Rockefeller University, 1230 York Avenue, New York, NY 10065, USA. Electronic address:

Neurons receive input from the outside world or from other neurons through neuronal receptive endings (NREs). Glia envelop NREs to create specialized microenvironments; however, glial functions at these sites are poorly understood. Here, we report a molecular mechanism by which glia control NRE shape and associated animal behavior. The C. elegans AMsh glial cell ensheathes the NREs of 12 neurons, including the thermosensory neuron AFD. KCC-3, a K/Cl transporter, localizes specifically to a glial microdomain surrounding AFD receptive ending microvilli, where it regulates K(+) and Cl(-) levels. We find that Cl(-) ions function as direct inhibitors of an NRE-localized receptor-guanylyl-cyclase, GCY-8, which synthesizes cyclic guanosine monophosphate (cGMP). High cGMP mediates the effects of glial KCC-3 on AFD shape by antagonizing the actin regulator WSP-1/NWASP. Components of this pathway are broadly expressed throughout the nervous system, suggesting that ionic regulation of the NRE microenvironment may be a conserved mechanism by which glia control neuron shape and function.
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http://dx.doi.org/10.1016/j.cell.2016.03.026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4860081PMC
May 2016

HSF-1 activates the ubiquitin proteasome system to promote non-apoptotic developmental cell death in C. elegans.

Elife 2016 Mar 8;5. Epub 2016 Mar 8.

Laboratory of Developmental Genetics, The Rockefeller University, New York, United States.

Apoptosis is a prominent metazoan cell death form. Yet, mutations in apoptosis regulators cause only minor defects in vertebrate development, suggesting that another developmental cell death mechanism exists. While some non-apoptotic programs have been molecularly characterized, none appear to control developmental cell culling. Linker-cell-type death (LCD) is a morphologically conserved non-apoptotic cell death process operating in Caenorhabditis elegans and vertebrate development, and is therefore a compelling candidate process complementing apoptosis. However, the details of LCD execution are not known. Here we delineate a molecular-genetic pathway governing LCD in C. elegans. Redundant activities of antagonistic Wnt signals, a temporal control pathway, and mitogen-activated protein kinase kinase signaling control heat shock factor 1 (HSF-1), a conserved stress-activated transcription factor. Rather than protecting cells, HSF-1 promotes their demise by activating components of the ubiquitin proteasome system, including the E2 ligase LET-70/UBE2D2 functioning with E3 components CUL-3, RBX-1, BTBD-2, and SIAH-1. Our studies uncover design similarities between LCD and developmental apoptosis, and provide testable predictions for analyzing LCD in vertebrates.
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http://dx.doi.org/10.7554/eLife.12821DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4821803PMC
March 2016

Cell Death in C. elegans Development.

Curr Top Dev Biol 2015 9;114:1-42. Epub 2015 Sep 9.

Laboratory of Developmental Genetics, The Rockefeller University, New York, USA. Electronic address:

Cell death is a common and important feature of animal development, and cell death defects underlie many human disease states. The nematode Caenorhabditis elegans has proven fertile ground for uncovering molecular and cellular processes controlling programmed cell death. A core pathway consisting of the conserved proteins EGL-1/BH3-only, CED-9/BCL2, CED-4/APAF1, and CED-3/caspase promotes most cell death in the nematode, and a conserved set of proteins ensures the engulfment and degradation of dying cells. Multiple regulatory pathways control cell death onset in C. elegans, and many reveal similarities with tumor formation pathways in mammals, supporting the idea that cell death plays key roles in malignant progression. Nonetheless, a number of observations suggest that our understanding of developmental cell death in C. elegans is incomplete. The interaction between dying and engulfing cells seems to be more complex than originally appreciated, and it appears that key aspects of cell death initiation are not fully understood. It has also become apparent that the conserved apoptotic pathway is dispensable for the demise of the C. elegans linker cell, leading to the discovery of a previously unexplored gene program promoting cell death. Here, we review studies that formed the foundation of cell death research in C. elegans and describe new observations that expand, and in some cases remodel, this edifice. We raise the possibility that, in some cells, more than one death program may be needed to ensure cell death fidelity.
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http://dx.doi.org/10.1016/bs.ctdb.2015.07.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5206663PMC
July 2016

FBN-1, a fibrillin-related protein, is required for resistance of the epidermis to mechanical deformation during C. elegans embryogenesis.

Elife 2015 Mar 23;4. Epub 2015 Mar 23.

Department of Molecular Biology, University of Wyoming, Laramie, United States.

During development, biomechanical forces contour the body and provide shape to internal organs. Using genetic and molecular approaches in combination with a FRET-based tension sensor, we characterized a pulling force exerted by the elongating pharynx (foregut) on the anterior epidermis during C. elegans embryogenesis. Resistance of the epidermis to this force and to actomyosin-based circumferential constricting forces is mediated by FBN-1, a ZP domain protein related to vertebrate fibrillins. fbn-1 was required specifically within the epidermis and FBN-1 was expressed in epidermal cells and secreted to the apical surface as a putative component of the embryonic sheath. Tiling array studies indicated that fbn-1 mRNA processing requires the conserved alternative splicing factor MEC-8/RBPMS. The conserved SYM-3/FAM102A and SYM-4/WDR44 proteins, which are linked to protein trafficking, function as additional components of this network. Our studies demonstrate the importance of the apical extracellular matrix in preventing mechanical deformation of the epidermis during development.
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http://dx.doi.org/10.7554/eLife.06565DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4395870PMC
March 2015

Glial development and function in the nervous system of Caenorhabditis elegans.

Authors:
Shai Shaham

Cold Spring Harb Perspect Biol 2015 Jan 8;7(4):a020578. Epub 2015 Jan 8.

Laboratory of Developmental Genetics, The Rockefeller University, New York, New York 10065.

The nematode, Caenorhabditis elegans, has served as a fruitful setting for understanding conserved biological processes. The past decade has seen the rise of this model organism as an important tool for uncovering the mysteries of the glial cell, which partners with neurons to generate a functioning nervous system in all animals. C. elegans affords unparalleled single-cell resolution in vivo in examining glia-neuron interactions, and similarities between C. elegans and vertebrate glia suggest that lessons learned from this nematode are likely to have general implications. Here, I summarize what has been gleaned over the past decade since C. elegans glia research became a concerted area of focus. Studies have revealed that glia are essential elements of a functioning C. elegans nervous system and play key roles in its development. Importantly, glial influence on neuronal function appears to be dynamic. Key questions for the field to address in the near- and long-term have emerged, and these are discussed within.
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http://dx.doi.org/10.1101/cshperspect.a020578DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4382739PMC
January 2015