Apoptotic and Nonapoptotic Cell Death in Caenorhabditis elegans Development.

Programmed cell death (PCD) is an essential component of animal development, and aberrant cell death underlies many disorders. Understanding mechanisms that govern PCD during development can provide insight into cell death programs that are disrupted in disease. Key steps mediating apoptosis, a highly conserved cell death program employing caspase proteases, were first uncovered in the nematode Caenorhabditis elegans, a powerful model system for PCD research. Recent studies in C. elegans also unearthed conserved nonapoptotic caspase-independent cell death programs that function during development. Here, we discuss recent advances in understanding cell death during C. elegans development. We review insights expanding the molecular palette behind the execution of apoptotic and nonapoptotic cell death, as well as new discoveries revealing the mechanistic underpinnings of dying cell engulfment and clearance. A number of open questions are also discussed that will continue to propel the field over the coming years.

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

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.

LET-381/FoxF and its target UNC-30/Pitx2 specify and maintain the molecular identity of C. elegans mesodermal glia that regulate motor behavior.

While most glial cell types in the central nervous system (CNS) arise from neuroectodermal progenitors, some, like microglia, are mesodermally derived. To understand mesodermal glia development and function, we investigated C. elegans GLR glia, which envelop the brain neuropil and separate it from the circulatory system cavity. Transcriptome analysis shows that GLR glia combine astrocytic and endothelial characteristics, which are relegated to separate cell types in vertebrates. Combined fate acquisition is orchestrated by LET-381/FoxF, a fate-specification/maintenance transcription factor also expressed in glia and endothelia of other animals. Among LET-381/FoxF targets, the UNC-30/Pitx2 transcription factor controls GLR glia morphology and represses alternative mesodermal fates. LET-381 and UNC-30 co-expression in naive cells is sufficient for GLR glia gene expression. GLR glia inactivation by ablation or let-381 mutation disrupts locomotory behavior and promotes salt-induced paralysis, suggesting brain-neuropil activity dysregulation. Our studies uncover mechanisms of mesodermal glia development and show that like neuronal differentiation, glia differentiation requires autoregulatory terminal selector genes that define and maintain the glial fate.

Glia-Neuron Interactions in Caenorhabditis elegans.

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 Caenorhabditis elegans 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 C. elegans. We highlight similarities with glia in other animals, and suggest conserved emerging principles of glial function.

Lineage-specific control of convergent differentiation by a Forkhead repressor.

During convergent differentiation, multiple developmental lineages produce a highly similar or identical cell type. However, few molecular players that drive convergent differentiation are known. Here, we show that the C. elegans Forkhead transcription factor UNC-130 is required in only one of three convergent lineages that produce the same glial cell type. UNC-130 acts transiently as a repressor in progenitors and newly-born terminal cells to allow the proper specification of cells related by lineage rather than by cell type or function. Specification defects correlate with UNC-130:DNA binding, and UNC-130 can be functionally replaced by its human homolog, the neural crest lineage determinant FoxD3. We propose that, in contrast to terminal selectors that activate cell type-specific transcriptional programs in terminally differentiating cells, UNC-130 acts early and specifically in one convergent lineage to produce a cell type that also arises from molecularly distinct progenitors in other lineages.

BLMP-1 promotes developmental cell death in C. elegans by timely repression of ced-9 transcription.

Programmed cell death (PCD) is a common cell fate in metazoan development. PCD effectors are extensively studied, but how they are temporally regulated is less understood. Here, we report a mechanism controlling tail-spike cell death onset during Caenorhabditis elegans development. We show that the zinc-finger transcription factor BLMP-1, which controls larval development timing, also regulates embryonic tail-spike cell death initiation. BLMP-1 functions upstream of CED-9 and in parallel to DRE-1, another CED-9 and tail-spike cell death regulator. BLMP-1 expression is detected in the tail-spike cell shortly after the cell is born, and blmp-1 mutations promote ced-9-dependent tail-spike cell survival. BLMP-1 binds ced-9 gene regulatory sequences, and inhibits ced-9 transcription just before cell-death onset. BLMP-1 and DRE-1 function together to regulate developmental timing, and their mammalian homologs regulate B-lymphocyte fate. Our results, therefore, identify roles for developmental timing genes in cell-death initiation, and suggest conservation of these functions.

DEX-1 and DYF-7 establish sensory dendrite length by anchoring dendritic tips during cell migration.

Cells are devices whose structures delimit function. For example, in the nervous system, neuronal and glial shapes dictate paths of information flow. To understand how cells acquire their shapes, we examined the formation of a sense organ in C. elegans. Using time-lapse imaging, we found that sensory dendrites form by stationary anchoring of dendritic tips during cell-body migration. A genetic screen identified DEX-1 and DYF-7, extracellular proteins required for dendritic tip anchoring, which act cooperatively at the time and place of anchoring. DEX-1 and DYF-7 contain, respectively, zonadhesin and zona pellucida domains, and DYF-7 self-associates into multimers important for anchoring. Thus, unlike other dendrites, amphid dendritic tips are positioned by DEX-1 and DYF-7 without the need for long-range guidance cues. In sequence and function, DEX-1 and DYF-7 resemble tectorins, which anchor stereocilia in the inner ear, suggesting that a sensory dendrite anchor may have evolved into part of a mechanosensor.

Cell death in animal development.

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.

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

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.

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

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.

Related F-box proteins control cell death in Caenorhabditis elegans and human lymphoma.

Cell death is a common metazoan cell fate, and its inactivation is central to human malignancy. In Caenorhabditis elegans, apoptotic cell death occurs via the activation of the caspase CED-3 following binding of the EGL-1/BH3-only protein to the antiapoptotic CED-9/BCL2 protein. Here we report a major alternative mechanism for caspase activation in vivo involving the F-box protein DRE-1. DRE-1 functions in parallel to EGL-1, requires CED-9 for activity, and binds to CED-9, suggesting that DRE-1 promotes apoptosis by inactivating CED-9. FBXO10, a human protein related to DRE-1, binds BCL2 and promotes its degradation, thereby initiating cell death. Moreover, some human diffuse large B-cell lymphomas have inactivating mutations in FBXO10 or express FBXO10 at low levels. Our results suggest that DRE-1/FBXO10 is a conserved regulator of apoptosis.

Chemosensory organs as models of neuronal synapses.

Neuronal synapses are important microstructures that underlie complex cognitive capacities. Recent studies, primarily in Caenorhabditis elegans and Drosophila melanogaster, have revealed surprising parallels between these synapses and the 'chemosensory synapses' that reside at the tips of chemosensory cells that respond to environmental stimuli. Similarities in the structures, mechanisms of action and specific molecules found at these sites extend to the presynaptic, postsynaptic and glial entities composing both synapse types. In this article I propose that chemosensory synapses may serve as useful models of neuronal synapses, and consider the possibility that the two synapse types derive from a common ancestral structure.

Two novel DEG/ENaC channel subunits expressed in glia are needed for nose-touch sensitivity in Caenorhabditis elegans.

Neuronal DEG/ENaC (degenerin and epithelial Na(+) channel) Na(+) channels have been implicated in touch sensation. For example, MEC-4 is expressed in touch neurons in Caenorhabditis elegans and mediates gentle-touch response. Similarly, homologous mammalian ASIC2 and ASIC3 are expressed in sensory neurons and produce touch phenotypes when knocked out in mice. Here, we show that novel DEG/ENaC subunits DELM-1 and DELM-2 (degenerin-like channel mechanosensory linked-1 and degenerin-like channel mechanosensory linked-2) are expressed in glia associated with touch neurons in C. elegans and that their knock-out causes defects in mechanosensory behaviors related to nose touch and foraging, which are mediated by OLQ and IL1 sensory neurons. Cell-specific rescue supports that DELM-1 and DELM-2 are required cell-autonomously in glia to orchestrate mechanosensory behaviors. Electron microscopy reveals that in delm-1 knock-outs, OLQ and IL1 sensory neurons and associated glia are structurally normal. Furthermore, we show that knock-out of DELM-1 and DELM-2 does not disrupt the expression or cellular localization of TRPA-1, a TRP channel needed in OLQ and IL1 neurons for touch behaviors. Rather, rescue of the delm-1 nose-touch-insensitive phenotype by expression of a K(+) channel in socket glia and of a cationic channel in OLQ neurons suggests that DELM channels set basal neuronal excitability. Together, our data show that DELM-1 and DELM-2 are expressed in glia associated with touch neurons where they are not needed for neuronal structural integrity or cellular distribution of neuronal sensory channels, but rather for their function.

A spatial and temporal map of C. elegans gene expression.

The C. elegans genome has been completely sequenced, and the developmental anatomy of this model organism is described at single-cell resolution. Here we utilize strategies that exploit this precisely defined architecture to link gene expression to cell type. We obtained RNAs from specific cells and from each developmental stage using tissue-specific promoters to mark cells for isolation by FACS or for mRNA extraction by the mRNA-tagging method. We then generated gene expression profiles of more than 30 different cells and developmental stages using tiling arrays. Machine-learning-based analysis detected transcripts corresponding to established gene models and revealed novel transcriptionally active regions (TARs) in noncoding domains that comprise at least 10% of the total C. elegans genome. Our results show that about 75% of transcripts with detectable expression are differentially expressed among developmental stages and across cell types. Examination of known tissue- and cell-specific transcripts validates these data sets and suggests that newly identified TARs may exercise cell-specific functions. Additionally, we used self-organizing maps to define groups of coregulated transcripts and applied regulatory element analysis to identify known transcription factor- and miRNA-binding sites, as well as novel motifs that likely function to control subsets of these genes. By using cell-specific, whole-genome profiling strategies, we have detected a large number of novel transcripts and produced high-resolution gene expression maps that provide a basis for establishing the roles of individual genes in cellular differentiation.

Glia delimit shape changes of sensory neuron receptive endings in C. elegans.

Neuronal receptive endings, such as dendritic spines and sensory protrusions, are structurally remodeled by experience. How receptive endings acquire their remodeled shapes is not well understood. In response to environmental stressors, the nematode Caenorhabditis elegans enters a diapause state, termed dauer, which is accompanied by remodeling of sensory neuron receptive endings. Here, we demonstrate that sensory receptive endings of the AWC neurons in dauers remodel in the confines of a compartment defined by the amphid sheath (AMsh) glial cell that envelops these endings. AMsh glia remodel concomitantly with and independently of AWC receptive endings to delimit AWC receptive ending growth. Remodeling of AMsh glia requires the OTD/OTX transcription factor TTX-1, the fusogen AFF-1 and probably the vascular endothelial growth factor (VEGFR)-related protein VER-1, all acting within the glial cell. ver-1 expression requires direct binding of TTX-1 to ver-1 regulatory sequences, and is induced in dauers and at high temperatures. Our results demonstrate that stimulus-induced changes in glial compartment size provide spatial constraints on neuronal receptive ending growth.

Opposing activities of LIT-1/NLK and DAF-6/patched-related direct sensory compartment morphogenesis in C. elegans.

Glial cells surround neuronal endings to create enclosed compartments required for neuronal function. This architecture is seen at excitatory synapses and at sensory neuron receptive endings. Despite the prevalence and importance of these compartments, how they form is not known. We used the main sensory organ of C. elegans, the amphid, to investigate this issue. daf-6/Patched-related is a glia-expressed gene previously implicated in amphid sensory compartment morphogenesis. By comparing time series of electron-microscopy (EM) reconstructions of wild-type and daf-6 mutant embryos, we show that daf-6 acts to restrict compartment size. From a genetic screen, we found that mutations in the gene lit-1/Nemo-like kinase (NLK) suppress daf-6. EM and genetic studies demonstrate that lit-1 acts within glia, in counterbalance to daf-6, to promote sensory compartment expansion. Although LIT-1 has been shown to regulate Wnt signaling, our genetic studies demonstrate a novel, Wnt-independent role for LIT-1 in sensory compartment size control. The LIT-1 activator MOM-4/TAK1 is also important for compartment morphogenesis and both proteins line the glial sensory compartment. LIT-1 compartment localization is important for its function and requires neuronal signals. Furthermore, the conserved LIT-1 C-terminus is necessary and sufficient for this localization. Two-hybrid and co-immunoprecipitation studies demonstrate that the LIT-1 C-terminus binds both actin and the Wiskott-Aldrich syndrome protein (WASP), an actin regulator. We use fluorescence light microscopy and fluorescence EM methodology to show that actin is highly enriched around the amphid sensory compartment. Finally, our genetic studies demonstrate that WASP is important for compartment expansion and functions in the same pathway as LIT-1. The studies presented here uncover a novel, Wnt-independent role for the conserved Nemo-like kinase LIT-1 in controlling cell morphogenesis in conjunction with the actin cytoskeleton. Our results suggest that the opposing daf-6 and lit-1 glial pathways act together to control sensory compartment size.

Glia Development and Function in the Nematode Caenorhabditis elegans.

The nematode Caenorhabditis elegans is a powerful experimental setting for uncovering fundamental tenets of nervous system organization and function. Its nearly invariant and simple anatomy, coupled with a plethora of methodologies for interrogating single-gene functions at single-cell resolution in vivo, have led to exciting discoveries in glial cell biology and mechanisms of glia-neuron interactions. Findings over the last two decades reinforce the idea that insights from C. elegans can inform our understanding of glial operating principles in other species. Here, we summarize the current state-of-the-art, and describe mechanistic insights that have emerged from a concerted effort to understand C. elegans glia. The remarkable acceleration in the pace of discovery in recent years paints a portrait of striking molecular complexity, exquisite specificity, and functional heterogeneity among glia. Glial cells affect nearly every aspect of nervous system development and function, from generating neurons, to promoting neurite formation, to animal behavior, and to whole-animal traits, including longevity. We discuss emerging questions where C. elegans is poised to fill critical knowledge gaps in our understanding of glia biology.

Some, but not all, retromer components promote morphogenesis of C. elegans sensory compartments.

The endings of sensory receptor cells often lie within specialized compartments formed by glial cells. The main sensory organ of Caenorhabditis elegans, the amphid, provides a powerful setting for studying glial compartment morphogenesis. Our previous studies showed that amphid compartment size is controlled by opposing activities of the Nemo-like kinase LIT-1, which promotes compartment expansion, and the Patched-related protein DAF-6, which restricts compartment growth. From a genetic screen for mutations able to suppress the bloated sensory compartments of daf-6 mutants, we identified an allele of the sorting nexin gene snx-1. SNX-1 protein is a component of the retromer, a protein complex that facilitates recycling of transmembrane proteins from the endosome to the Golgi network. We find that snx-1 functions cell autonomously within glia to promote sensory compartment growth, and that SNX-1 protein is enriched near the surface of the sensory compartment. snx-1 interacts genetically with lit-1 and another regulator of compartment size, the Dispatched-related gene che-14. Mutations in snx-3 and vps-29, also retromer genes, can suppress daf-6 defects. Surprisingly, however, remaining retromer components seem not to be involved. Our results suggest that a novel assembly of retromer components is important for determining sensory compartment dimensions.

Shai Shaham.

Interview with Shai Shaham, who studies glia-neuron interactions in C. elegans at The Rockefeller University.

LET-381/FoxF and UNC-30/Pitx2 control the development of C. elegans mesodermal glia that regulate motor behavior.

While most CNS glia arise from neuroectodermal progenitors, some, like microglia, are mesodermally derived. To understand mesodermal glia development and function, we investigated C. elegans GLR glia, which ensheath the brain neuropil and separate it from the circulatory-system cavity. Transcriptome analysis suggests GLR glia merge astrocytic and endothelial characteristics relegated to separate cell types in vertebrates. Combined fate acquisition is orchestrated by LET-381/FoxF, a fate-specification/maintenance transcription factor expressed in glia and endothelia of other animals. Among LET-381/FoxF targets, UNC-30/Pitx2 transcription factor controls GLR glia morphology and represses alternative mesodermal fates. LET-381 and UNC-30 co-expression in naïve cells is sufficient for GLR glia gene expression. GLR glia inactivation by ablation or let-381 mutation disrupts locomotory behavior and induces salt hypersensitivity, suggesting brain-neuropil activity dysregulation. Our studies uncover mechanisms of mesodermal glia development and show that like neurons, glia differentiation requires autoregulatory terminal selector genes that define and maintain the glial fate.