Shaham S (2023) Curr Biol "Shai Shaham."

Shaham S

[Curr Biol, 2023]

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

Shai Shaham (2006) "Methods in cell biology"

Shai Shaham

[2006]

Shai Shaham et al. (2001) International C. elegans Meeting "The cfi-1 gene inhibits expression of CEM cell fate in other C. elegans neurons."

Shai Shaham, Cori Bargmann

[International C. elegans Meeting, 2001]

We are interested in understanding cues that determine cell fate in C. elegans . Specifically, we aim to understand how cell fate determinants activate or inhibit programmed cell death (PCD). To this end we have been studying cues affecting differentiation and sex-specific survival of the male-specific CEM neurons. We showed that CEM death requires the cell death genes egl-1 , ced-3 and ced-4 and is inhibited by the cell death gene ced-9 . Mutations in tra-2 and tra-1 , which promote male identity in XX animals, prevent CEM death. We also isolated mutants in which CEM neurons expressing a pkd-2 ::GFP transgene (from M. Barr and P. Sternberg) survive inappropriately in hermaphrodites. From a screen of 9500 genomes we identified 25 independent mutants. 15 specifically affect survival of the CEMs and no other discernable aspect of development. Five contain mutations in general programmed cell death genes and three affect sex determination. The remaining two mutants fail to complement and result in the transformation of some head neurons (probably the IL2s) into CEM-like neurons. Transformed cells express CEM-specific markers, exhibit a nuclear morphology unique to the CEMs, and their cell bodies are sometimes located where CEMs' normally reside. We have named the gene affected in these mutants cfi-1 (for CEM fate inhibitor). The CEMs die appropriately in these mutants, but ectopic CEM-like neurons survive in both sexes, suggesting that neuronal identity and survival are separable aspects of CEM fate. To understand the molecular mechanism underlying cfi-1 function we are cloning the gene. We have mapped the gene to a 0.5 map unit interval on chromosome I, and have obtained rescue using a single cosmid. Our results will be presented at the meeting. To further understand determinants that regulate CEM neuronal identity we have been studying regulation of pkd-2 ::GFP expression in these cells and in CEM-like neurons generated in cfi-1 mutants. CEM-like cells express pkd-2 ::GFP at higher levels in male than in hermaphrodite cfi-1 mutants. Similarly, pkd-2 ::GFP expression in CEMs is stronger in male vs. hermaphrodite ced-3 mutants. Thus, the sex determination pathway can impinge on CEM development at several levels; by regulating PCD and by regulating cell-type specific gene expression. Mutations in either lin-32 , which encodes a bHLH transcription factor, or unc-86 , a POU-domain transcription factor, strongly inhibit pkd-2 ::GFP expression in CEM-like cells in cfi-1 hermaphrodites and in normal CEMs in males. These results suggest that pkd-2 ::GFP expression, and perhaps CEM neuronal identity, are promoted by the absence of cfi-1 activity, presence of lin-32 and unc-86 activities, and male sexual identity.

Shaham S et al. (1992) Worm Breeder's Gazette "Molecular Analysis of the Cell Death Gene ced-3"

Horvitz HR, Shaham S

[Worm Breeder's Gazette, 1992]

MOLECULAR ANALYSIS OF THE CELL DEATH GENE ced-3 Shai Shaham and Bob Horvitz ~lMI, Dept. Biology, MIT, Cambridge, MA 02139

Shai Shaham et al. (2002) East Coast Worm Meeting "daf-6, a gene regulating morphology of the glial-like amphid sheath cells, encodes a Patched-related protein."

Mark Schroeder, Shai Shaham, Elliot Perens, Max Heiman

[East Coast Worm Meeting, 2002]

Glia account for over 90% of the cells populating the human brain, and glia-derived tumors are the most common and most lethal human brain tumors. Surprisingly, compared to their neuronal counterparts, little is known about glial cell development, function, or morphogenesis. Growing evidence suggests that glia play active roles in regulating synaptic transmission and neuronal current propagation, and are likely to play key roles in many, if not all, aspects of nervous system function.

Shai Shaham et al. (2002) East Coast Worm Meeting "CED-13, a novel BH3 domain protein, regulates radiation-induced and developmental programmed cell death in C. elegans."

Simon Tuck, Nicole Wittenburg, BARBARA CONRADT, Bjorn Schumacher, Anton Gartner, Shai Shaham

[East Coast Worm Meeting, 2002]

Programmed cell death (PCD) is a common cell fate in the development of multicellular organisms. In C. elegans, for example, 131 of the 1090 somatic cells that are born undergo PCD, and nearly half of the cells born in the C. elegans germline have a similar fate. Although much has been learned about the machinery that executes PCD in C.elegans and in mammals, only in a few examples have the signal transduction pathways leading to activation of this machinery been elucidated. Current models suggest that most, if not all PCDs are controlled by three main gene families. In C. elegansthese are represented by ced-9 (abcl-2 family member), ced-4 (similar to mammalian Apaf-1), and ced-3 (a caspase family member). These genes act in a pathway such that ced-9 inhibits ced-4, which in turn activates ced-3, promoting PCD. We are interested in defining molecular signaling events that trigger activation of this pathway. We have identified a novel gene, ced-13(formerly cip-1), whose protein product can interact with the CED-9 protein. We have shown that CED-13 protein binds to CED-9 in a two-hybrid assay. Co-precipitation experiments using a GST-CED-9 fusion protein and 35 S-labelled CED-13 also indicate that CED-9 and CED-13 may normally interact. Sequence inspection suggests that CED-13 contains a BH3 (bcl-2homology region 3) domain that may mediate interaction with CED-9, a hypothesis supported by our binding studies. The C. elegans BH3 domain protein EGL-1 interacts with CED-9 in a similar manner. To define the function of ced-13 we overexpressed the gene using C. elegans heat shock promoters. We found that embryos overexpressing ced-13 are dead. This inviability can be rescued by loss-of-function mutations in the ced-3, or ced-4genes, or by a gain-of-function mutation in ced-9. These observations suggest that CED-13 acts upstream of the core PCD machinery to promote PCD. We isolated a deletion allele of ced-13that lacks a 1.3 kb DNA segment containing the gene. ced-13 mutants are weakly defective in developmental PCD. Specifically, double mutants of ced-13 and a weak ced-3 mutation are more defective for PCD than mutants carrying either single mutation. More strikingly, however, whereas radiation normally induces many cell deaths in the germlines of wild-type adults, ced-13 mutants exhibit no radiation-induced PCD. These results support the notion that ced-13is an important activator of PCD in C. elegans. Our preliminary results suggest that ced-13 may be transcriptionally induced following irradiation, and that this induction may be dependent on the C. elegansp53-related gene cep-1. Thus, our studies suggest that a radiation-response signal transduction pathway impinges on ced-13 to regulate PCD in the germline. Interestingly, the egl-1 gene shows a partial defect in PCD following irradiation and a severe defect in developmental PCD. We propose that egl-1 and ced-13 together regulate both types of death.

Zuckerman, Jennifer et al. (2010) C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany "Uncovering the role of LET-70 in linker cell death in C. elegans"

Zuckerman, Jennifer, Shaham, Shai

[C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany, 2010]

Cell death is an essential process, providing a means to direct tissue morphogenesis. The C. elegans linker cell is an important regulator of male gonad morphogenesis. It directs migration of the gonad, and its death allows fusion of the vas deferens with the cloacal exit channel. Unlike most dying cells, which die shortly after they are generated, the linker cell survives for two days before its death during the L4/adult transition. Studies from our lab showed that linker cell death is independent of known C. elegans cell death genes and is characterized by nonapoptotic features, including nuclear membrane invagination and lack of chromatin condensation. These features have been described in cell death that accompanies normal development of the vertebrate nervous system and in neurodegenerative diseases promoted by p olyglutamine expansions. Interestingly, an RNAi screen for genes important in linker cell death identified the gene pqn-41, a polyglutamine repeat protein, as a regulator of linker cell death (E Blum and S Shaham, unpublished). Additional genes were also identified, including let-70 , a putative E2 ubiquitin-conjugating enzyme. Wild-type males fed E. coli expressing let-70 dsRNA have surviving linker cells. A block in the final stage of linker cell migration is also seen, but is separable from the death defect. The let-70 defect is not enhanced by mutations in sek-1 /MAPKK but is slightly enhanced by mutations in the Zn-finger transcriptional regulator lin-29 . sek-1 and lin-29 seem to act in parallel to promote linker cell death. Linker cell survival is enhanced by a mutation in pqn-41 . Like pqn-41 , expression of let-70 promoter::GFP transgenes is induced in the linker cell as it begins to die. Several studies suggest that polyglutamine proteins promoting neuodegeneration are ubiquitinated. We are interested in testing the possibility that PQN-41 or other proteins implicated in linker cell death are modified by LET-70.

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.

Singhal, Anupriya et al. (2015) International Worm Meeting "Cell-Specific Labeling of Neurons and Glia in the C. elegans embryo using Infrared Laser Illumination."

Singhal, Anupriya, Shaham, Shai

[International Worm Meeting, 2015]

Glial cells play critical roles in the development of the nervous system in C. elegans, but neuron-glia interactions during embryogenesis and intermediate stages of nervous system assembly are poorly understood. A major challenge in studying development of the embryonic nervous system is that most cell-specific reporters are not expressed early enough in embryogenesis to visualize the early stages of neurite outgrowth. To label neural and glial precursors in the embryo specifically, we are developing a method, based on a previously described setup (Kamei et al, 2009), which uses an infrared laser to localize the heat shock response to individual cells and labels them via a heat shock-driven transgene. We have adapted this method for use in embryonic cells, which are smaller than larval cells and more sensitive to heat toxicity, by using ratiometric fluorescent reporters to measure the temperature induced by the laser directly in vivo. Using our optimized method, cells are heated to physiological heat shock temperatures (32oC) for 5-10 minute durations, leading to high rates of induction (>50%) with no observable damage or off-target induction. Using our method, we are able to achieve cell-specific labeling of neuronal and glial precursors at the 100-200 cell stage, after which the daughters are labeled with a constitutive reporter generated by Cre-Lox recombination. With time-lapse imaging, we can observe dendrite extension and axon outgrowth of labeled cells during nervous system development. Heat-shock labeled cells produce complete axon and dendrite trajectories and subsequently express post-embryonic fate markers, indicating that development after heat shock proceeds normally. We are currently extending this method for use in embryonic cell-specific rescues. We plan to apply this method to study the morphogenesis of neuron and glial cells in quantitative fashion during the development of the C. elegans nervous system.

Wallace, Sean et al. (2021) International Worm Meeting "Nuclear hormone receptors mediate adaptive responses to the mold Penicillium brevicompactum"

Shaham, Shai, Wallace, Sean

[International Worm Meeting, 2021]

Xenobiotic metabolizing enzymes (XMEs) play a major role in detoxification. Previous studies of XMEs in C. elegans have primarily revealed a role in the gut. By carrying out transcriptome analysis of amphid sheath (AMsh) glial cells, we have found many detoxifying enzymes to be expressed in glia, including cytochrome P450 oxygenases (CYPs), UDP-glucoronosyltransferases (UGTs) and glutathione-S-transferases (GSTs). Intriguingly, high expression levels of XMEs are also observed in the sustentacular glial cells of the mammalian olfactory epithelium, the analogous cell-type to AMsh glia, but the functions of these enzymes in olfactory tissues are poorly characterized. We have found that expression of the cytochrome P450 gene cyp-33C2, in both glia and gut, is induced by exposure to Penicillium brevicompactum, a toxin-producing mold. Penicillium species are common laboratory contaminants, and they are also found naturally in environments that wild isolates of C. elegans inhabit, yet there have been surprisingly few studies exploring the interactions between C. elegans and Penicillium species. We have found that the nuclear hormone receptors nhr-45 and nhr-156 are required cell-autonomously for mold-dependent induction of cyp-33C2. nhr-45 has been previously implicated in mitochondrial stress induced by the pathogen Pseudomonas aeruginosa. We have found that the mold P. brevicompactum causes mitochondrial stress in the gut and that this is greatly enhanced in nhr-45 mutant animals that are unable to induce expression of detoxifying enzymes. This elevated mitochondrial stress is accompanied by severe developmental defects, which can be rescued by restoring nhr-45 expression in the gut. These findings suggest that nuclear hormone receptor-dependent expression of detoxifying enzymes in the gut is required to clear mold toxins. We are currently exploring similar mechanisms in AMsh glia. The nuclear hormone receptor family has undergone massive expansion and diversification in the C. elegans genome. It has been proposed that this expansion allows NHRs to act as receptors for varying environmental cues. Intriguingly, naturally occurring polymorphisms in the nhr-156 gene in wild isolates of C. elegans, including the Hawaiian isolate CB4856, result in a reduced transcriptional response in AMsh glia upon mold exposure. Remarkably, using CRISPR to substitute the nhr-156 locus from CB4856 in to the N2 background is sufficient to generate the observed phenotypic diversity. While the implications of these findings are still under investigation, they support the idea that diversification of nuclear hormone receptor sequences in C. elegans allows for adaptations to environmental conditions.