Nakano, Shunji et al. (2009) International Worm Meeting "ngn-1 and hlh-2 Are Required for a Left-Right Asymmetric Neurogenesis Decision."

Nakano, Shunji, Ellis, Ronald, Horvitz, Bob

[International Worm Meeting, 2009]

The body plan of C. elegans is mostly bilaterally symmetric. Much of this symmetry arises from analogous blastomeres, which through bilaterally symmetric cell lineages generate sets of left-right paired cells. To create either asymmetric cells or three-fold symmetry, this bilateral symmetry must be broken. The e3 pharyngeal epithelial cells form a three-fold symmetric structure that is composed of three cells, located on the ventral left (VL), ventral right (VR) and dorsal (D) region of the pharynx. e3VL and e3VR are generated as left-right lineal homologs, whereas e3D is generated by breaking left-right symmetry in a specific cell lineage. Specifically, the blastomeres ABaraapa and ABaraapp are sister cells that give rise to identical sets of left-right paired cells, except that the ABaraapaaaa cell becomes the e3D cell and its lineally homologous cell ABaraappaaa becomes the MI neuron. The differential determination of cell fate by these two cells breaks left-right symmetry in these cell lineages. We sought mutations that transform MI into an e3-like cell or e3D into an MI-like cell, thereby regenerating symmetry in these cell lineages. From screens of 10,000 mutagenized haploid genomes, we identified two genes, ngn-1 (neurogenin-1) and hlh-2 (helix-loop-helix-2), that when mutated cause the absence of MI and the presence of an extra e3-like cell. Laser ablation of the grandmother of the presumptive MI neuron in the ngn-1 mutant results in the absence of the extra e3-like cell. These results suggest that MI is transformed into an e3-like cell in the mutants. ngn-1 encodes a basic helix-loop-helix (bHLH) protein. hlh-2 is the C. elegans ortholog of E2A/Daughterless, a bHLH protein known to dimerize with other bHLH proteins, including those within the neurogenin subfamily. Mosaic analysis of ngn-1 suggests that ngn-1 acts cell-autonomously to specify the MI fate. We are currently examining the expression of ngn-1 and hlh-2 to test whether these genes are expressed asymmetrically to break the left-right symmetry in this cell lineage. In short, we have identified two bHLH genes required for breaking symmetry in a left-right asymmetric cell lineage. We hypothesize that NGN-1 and HLH-2 form a transcriptional heterodimer that acts cell autonomously to induce left-right asymmetric neurogenesis. We hope that further analysis of these genes will uncover a mechanism by which ngn-1 and hlh-2 are differentially regulated to result in an invariant determination of this aspect of left-right asymmetry.

Nakano, Shunji et al. (2013) International Worm Meeting "Identification of New Genes Involved in C. elegans Thermotaxis Behavior."

Nakano, Shunji, Mori, Ikue, Higashiyama, Tetsuya, Suzuki, Takamasa, Tianyu, Jiang

[International Worm Meeting, 2013]

C. elegans can sense and remember the environmental temperature and navigate themselves toward the cultivation temperature when placed on a thermal gradient. Although studies of the past decades have identified genes and neural circuits involved in thermotaxis, how C. elegans achieves this complex behavior still remains elusive. To further reveal the neural mechanisms that underlie thermotaxis, we undertook genetic screens to look for mutants defective in thermotaxis. We screened approximately 70,000 F2 progeny of mutagenized wild-type animals for mutants that displayed abnormal thermotaxis behavior and recovered 24 isolates. These 24 mutants can be classified into three phenotypic classes: 12 mutants are thermophilic, 4 mutants cryophilic and 8 mutants athermotactic. We began our analysis with the 12 thermophilic mutants. Based on complementation tests, we found that three of the 12 mutations are alleles of genes previously shown to be important for thermotaxis. nj98 and nj111 are alleles of ttx-4/pkc-1, and nj113 is an allele of tax-6. The remaining 9 mutations likely represent new genes involved in thermotaxis. We thus far identified that 5 mutations mapped to regions in which no gene previously shown to regulate thermotaxis is present. nj104 and nj108 failed to complement each other and mapped to a 178 kb region of LG X. nj97 mapped to a 1.9 Mb region of LG X that excludes the nj104 and nj108 loci. nj100 mapped to a 600 kb region of LG II. nj102 mapped to a 308 kb region of LG IV. In addition, two mutations, nj89 and nj85, at least semi-dominantly cause thermophilic defects and mapped to LG X. In short, our screens recovered mutations that represent at least 4 novel genes important for thermotaxis. We are currently performing whole-genome sequencing of these mutants to identify the genes mutated in these isolates. Identification and characterization of these new genes will further reveal the neural mechanisms that generate thermotaxis behavior.

Nakano, Shunji et al. (2015) International Worm Meeting "The C. elegans MAST Kinase Acts through Stomatin to Regulate Thermotaxis Behavior."

Sano, Ayana, Nakano, Shunji, Mori, Ikue, Ikeda, Muneki, Higashiyama, Tetsuya, Suzuki, Takamasa, C. Giles, Andrew

[International Worm Meeting, 2015]

C. elegans can sense and remember the environmental temperature and navigate themselves toward that temperature when placed on a thermal gradient. Although previous studies identified genes and neural circuits involved in thermotaxis, how C. elegans achieves this complex behavior still remains elusive. To further reveal the mechanisms underlying thermotaxis, we undertook a genetic screen to look for mutants defective in thermotaxis. We found that a null mutation in the gene kin-4, which encodes the C. elegans homolog of MAST (Microtubule Associated Serine Threonine) kinase, caused a cryophilic phenotype. Expression of kin-4 in the AFD thermosensory neuron rescued this defect. We also found that nj89, another isolate recovered from our screen, caused a thermophilic defect and suppressed the kin-4 mutant phenotype. nj89 is a gain-of-function mutation of mec-2, which encodes a stomatin-like membrane-associated protein previously shown to bind to microtubules and a DEG/ENaC (Degenerin/Epithelial Na Channel). To identify the behavioral components defective in these mutants, we set up a high-throughput tracking system to record thermotaxis behavior. Wild-type animals modulate the frequencies of omega and reversal turns. They also bias the angle of shallow turns to steer toward the cultivation temperature. We found that kin-4 and mec-2 mutants are defective in regulating reversal turn frequencies . Our results suggest a novel signal transduction pathway in which KIN-4/MAST kinase regulates MEC-2/Stomatin. Since both a mammalian MAST kinase and MEC-2 are known to associate with microtubules, KIN-4 might bind to microtubules and regulate the activity of MEC-2. Furthermore, given that MEC-2 modulates ENaC activity and that some ENaCs respond to temperature change, the KIN-4/MEC-2 pathway might regulate ENaC activity and control a process in the AFD sensory neuron, such as temperature sensing. Further study will assess whether ENaCs act with KIN-4/MEC-2 during thermotaxis and identify the molecular and neural mechanisms by which KIN-4/MEC-2 controls thermotaxis behavior.

Nakano, Shunji et al. (2013) International Worm Meeting "Tousled-like Kinase Is Required to Generate a Bilateral Asymmetry in the C. elegans Nervous System."

Mori, Ikue, Nakano, Shunji, Horvitz, Bob

[International Worm Meeting, 2013]

Bilateral asymmetry in C. elegans can arise from left-right asymmetric cell lineages. The single left-right unpaired MI neuron descends from the right side of an otherwise left-right symmetric cell lineage: the homologous cell on the left side becomes the e3D epithelial cell. To identify the mechanisms that generate the MI-e3D asymmetry, we screened for mutants in which the MI-e3D asymmetry is lost. We isolated 16 mutations in 7 genes that transform MI into an e3D-like cell, thereby generating symmetry in a normally left-right asymmetric cell lineage. We previously showed that the establishment of the MI-e3D asymmetry requires left-right asymmetric expression of a CEH-36/NGN-1/HLH-2 transcriptional cascade (Nakano et al., Development, 2010). CEH-36 homeodomain protein is expressed in the MI grandmother cell but not in the e3D grandmother cell. CEH-36 induces asymmetric expression of two bHLH proteins, NGN-1 and HLH-2, in the MI mother cell but not in the e3D mother cell. We also showed that replication-coupled chromatin assembly is necessary for the MI-e3D asymmetry (Nakano et al., Cell, 2011). Reduction of the activity of the CAF-1 complex, a histone chaperone that deposits histone H3-H4 onto replicating DNA, causes the loss of the MI-e3D asymmetry. Here we report that two mutations isolated from our screens, n5342 and n5351, are alleles of the gene tlk-1. These mutant animals carry mutations in the tlk-1 locus. Animals carrying a tlk-1 deletion allele, tm2395, also show the loss of the MI-e3D asymmetry. tlk-1 encodes an evolutionarily conserved serine-threonine kinase implicated in several chromatin-based processes, including transcriptional regulation, chromosome segregation and DNA damage responses. We are planning to test whether tlk-1 is involved in CAF-1-mediated chromatin assembly or regulates expression of CEH-36, NGN-1 or HLH2. We hope that continued characterization of these genes will further reveal the mechanisms that establish bilateral asymmetry in the C. elegans nervous system.

Nakano, Shunji et al. (2011) International Worm Meeting "Replication-Coupled Nucleosome Assembly Is Required to Generate a Bilateral Asymmetry in the C. elegans Nervous System."

Stillman, Bruce, Horvitz, Bob, Nakano, Shunji

[International Worm Meeting, 2011]

Bilateral asymmetry in C. elegans can arise from left-right asymmetric cell lineages. The single left-right unpaired MI neuron descends from the right side of an otherwise left-right symmetric cell lineage that on the left generates the e3D epithelial cell. We sought mutations that transform MI into an e3D-like cell or e3D into an MI-like cell, thereby generating symmetry in these cell lineages. We recovered 16 mutations in 7 genes that transform MI into an e3D-like cell. We previously showed that the establishment of the MI-e3D asymmetry requires asymmetric expression of a transcriptional cascade in which the Otx homeodomain protein CEH-36 is expressed in the MI grandmother cell but not in the e3D grandmother cell and that CEH-36 promotes asymmetric expression of two bHLH proteins, NGN-1 and HLH-2, in the MI mother cell but not in the e3D mother cell (Nakano et al., Development 137, 4017, 2010). Here we show that another isolate, n5357, is a gain-of-function allele of the gene his-9, which encodes a replication-dependent histone H3 protein. This his-9(gf) mutation acts cell-autonomously in the MI mother cell and affects a process downstream of or in parallel to the expression of CEH-36, NGN-1 and HLH-2. Replication-dependent histone H3-H4 proteins are deposited onto nucleosomes by the CAF-1 complex during DNA replication. The human CAF-1 complex is composed of three subunits, p150, p60 and p48. Inactivation of T06D10.2, Y71G12B.1 and rba-1, which encode proteins homologous to p150, p60 and p48, respectively, transformed MI into an e3D-like cell. That a gain-of-function mutation in a histone H3 gene phenocopied the loss of CAF-1 function suggests that the mutant H3 protein inhibits CAF-1-mediated nucleosome formation. We performed a nucleosome assembly reaction to monitor CAF-1 activity in vitro and observed that corresponding mutant histone H3 proteins indeed inhibited CAF-1-mediated nucleosome formation. Our results reveal that replication-coupled nucleosome assembly is required to generate the MI-e3D asymmetry. We suggest that during S phase of the MI mother cell CAF-1 assembles nucleosomes on which the NGN-1/HLH-2 complex recruits histone-modifying enzymes to generate a chromatin state necessary to specify MI and that CAF-1-mediated nucleosome formation and the asymmetric localization of the NGN-1/HLH-2 complex drive left-right asymmetric epigenetic regulation, leading to the establishment of the MI-e3D asymmetry.

Shunji Nakano et al. (2007) International Worm Meeting "Mutations that Restore Symmetry in a Left-Right Asymmetric Cell Lineage."

Shunji Nakano, Bob Horvitz, Ronald E. Ellis, Victoria Hatch

[International Worm Meeting, 2007]

The body plan of Caenorhabditis elegans is mostly bilaterally symmetric. Much of the symmetry arises from pairs of bilaterally symmetric homologous blastomeres, which through similar cell lineages give rise to sets of left-right paired cells. To create either asymmetric cells or three-fold symmetry, the bilateral symmetry in cell lineages must be broken. The pharyngeal epithelial cells form a three-fold symmetric structure that consists of nine cells. The nine cells are divided into three groups of three cells, e1, e2, and e3, on the ventral left (VL), ventral right (VR), and dorsal (D) regions of the pharynx. e3VL and e3VR are generated as lineal homologs, whereas e3D is generated by breaking left-right symmetry in a specific cell lineage. The blastomeres ABaraapa and ABaraapp are homologous cells that give rise to identical sets of left-right paired cells, except that the ABaraapaaaa cell becomes the e3D cell and its lineally homologous cell ABaraappaaa becomes the MI neuron. The differential determination of cell fate by these two cells breaks left-right symmetry in these cell lineages. We sought mutations that transform MI into an e3-like cell or e3D into an MI-like cell, thereby regenerating symmetry in these cell lineages. From screens of 10,000 mutagenized haploid genomes and unrelated screens performed previously, we identified four independent mutations that cause transformation of MI into an e3-like cell fate.Three of the four mutations, n1921, n5020, and n5052, define a single complementation group. We have mapped n1921 to a 153 kb interval on LG IV. The other mutation, n5053, semidominantly transforms MI into an e3-like cell fate and maps to LG I. n5053 or a mutation closely linked to n5053 appears to recessively cause embryonic lethality. Thus, we have identified four mutations that restore symmetry in a left-right asymmetric cell lineage, and these mutations define at least two genes.

Shunji Nakano et al. (2006) Development & Evolution Meeting "Genetic Screens for Genes Involved in Left-Right Asymmetric Programmed Cell Death"

Shunji Nakano, Bob Horvitz

[Development & Evolution Meeting, 2006]

The body plan of Caenorhabditis elegans is mostly bilaterally symmetric. Much of the symmetry can be traced to a series of symmetric cell divisions of homologous blastomeres, which, through similar cell lineages, give rise to a set of left-right paired cells. To create an asymmetric cell, bilateral symmetry in cell lineages must be broken. The complete cell lineage of C. elegans reveals various mechanisms by which asymmetric cells are generated: one such mechanism involves a programmed cell death that eliminates one of two members of a left-right symmetrical pair. Two blastomeres, ABalapap and ABalappp, are homologous cells that give rise to identical sets of left-right paired cells except for two sister neurons, ALA and RMED, that are generated on only the right side as some of the descendants of ABalappp. On the left side, among the cells descended from ABalapap, the homologous cell of the mother of ALA and RMED (ABalapapaa) undergoes programmed cell death, thereby breaking left-right symmetry. We hope to investigate how this programmed cell death is specified and how this asymmetry in cell lineages is established. To this end, we are planning to perform two genetic screens. unc-25::gfp is expressed in all GABAergic neurons, including RMED. Using this reporter gene, we will screen for mutants lacking RMED, with a particular interest in mutants suppressible by a defect in programmed cell death. For the second screen, an ALA marker, ceh-17::gfp, will be used to look for mutants containing an extra ALA-like cell. Candidates from these screens might include mutants in which the asymmetric cell lineage becomes symmetric or specification of the programmed cell death is abnormal.

Shunji Nakano et al. (2008) Development & Evolution Meeting "Two bHLH Transcription Factors Are Required for Breaking Symmetry in a Left-Right Asymmetric Cell Lineage"

Ronald Ellis, Bob Horvitz, Shunji Nakano

[Development & Evolution Meeting, 2008]

The body plan of C. elegans is mostly bilaterally symmetric. Much of this symmetry arises from pairs of homologous blastomeres, which through bilaterally symmetric cell lineages give rise to sets of left-right paired cells. To create either asymmetry or three-fold symmetry, the bilateral symmetry in cell lineages must be broken. The pharyngeal epithelial cells form a three-fold symmetric structure that consists of nine cells. The nine cells are divided into three groups of three cells, e1, e2, and e3, on the ventral left (VL), ventral right (VR), and dorsal (D) regions of the pharynx. e3VL and e3VR are generated as lineal homologs, whereas e3D is generated by breaking left-right symmetry in a specific cell lineage.The blastomeres ABaraapa and ABaraapp are sister cells that give rise to identical sets of left-right paired cells, except that the ABaraapaaaa cell becomes the e3D cell and its lineally homologous cell ABaraappaaa becomes the MI neuron. The differential determination of cell fate by these two cells breaks left-right symmetry in these cell lineages. We sought mutations that transform MI into an e3-like cell or e3D into an MI-like cell, thereby regenerating symmetry in these cell lineages. From screens of 10,000 mutagenized haploid genomes and unrelated screens performed previously seeking mutants abnormal in pharyngeal anatomy, we identified four independent mutations that cause transformation of MI into an e3-like cell fate. Three of the four mutations, n1921, n5020, and n5052, define a single complementation group. The other mutation, n5053, semidominantly transforms MI into an e3-like cell fate and causes recessive embryonic lethality. n1921, n5020, and n5052 are alleles of ngn-1 (neurogenin-1), which encodes a basic helix-loop-helix (bHLH) protein. n5053 is an allele of hlh-2 (helix-loop-helix-2), the C. elegans ortholog of Drosophila E/ Daughterless. Daughterless encodes a bHLH protein known to dimerize with other bHLH proteins, including those within the neurogenin subfamily. RNAi of either ngn-1 or hlh-2 resulted in transformation of MI into an e3-like fate. Thus, we have identified two genes required for breaking symmetry in a left-right asymmetric cell lineage. We hypothesize that NGN-1 and HLH-2 form a transcriptional heterodimer required for establishing the asymmetry. We hope that further analysis of these genes will uncover a mechanism by which the invariant determination of left-right asymmetry is specified.

Ahluwalia, Rhea et al. (2021) International Worm Meeting "Discerning the Role of Neuropeptide(s) in C. elegans Thermotaxis Behavior"

Ahluwalia, Rhea, Mori, Ikue, Jang, Moon Sun, Nakano, Shunji

[International Worm Meeting, 2021]

The C. elegans genome encodes over a hundred and fifty neuropeptide genes, which consist of three classes and partake in various functions such as synaptic transmission, cellular signaling and development. Despite their ubiquitous nature, the role(s) played by neuropeptides in several behaviors in C. elegans remains elusive. One such behavioral paradigm is thermotaxis, a robust behavior wherein well-fed worms have been shown to migrate towards the region of past cultivation temperature when placed on a thermal gradient. Studies have suggested the possible involvement of neuropeptides in communication within the neural circuits required for thermotaxis, however, their exact role in thermotaxis regulation is unknown (Narayan, A., Laurent, G. and Sternberg, P. W. (2011) and Nakano, S. et al. (2020)). In order to confirm and further uncover how neuropeptide(s) might be partaking in thermotaxis, we must first identify which neuropeptide(s) are involved for this behavior. To this end, we are performing a genome-wide neuropeptide screen to look for neuropeptide candidates that may be involved in thermotaxis behavior, along with cell-specific knockout for genes crucial for neuropeptide processing in C. elegans. Thus far, we have tested over a hundred mutants, and none have shown strongly altered thermotaxis behavior. In addition, we have tested the four known pro-protein convertases in C. elegans and found that egl-3, the C. elegans ortholog of human proprotein convertase 2 (PCSK 2), displayed abnormal thermotaxis behavior. However, an AFD-specific knockout of egl-3 displayed thermotaxis behavior akin to wildtype, suggesting that egl-3 mediated regulation of thermotaxis most likely takes place through an alternative pathway, prompting further questions pertaining to site of action and the role of the neuropeptides processed by egl-3 in thermotaxis behavior. Altogether, through the genome-wide neuropeptide screen along with the study of the neuropeptide processing enzymes, we hope to gain a holistic insight into peptidergic modulation that controls thermotaxis behavior.

Tsukamoto, Satomi et al. (2013) International Worm Meeting "The roles of biogenic amines on feeding state-dependent thermotactic behavior in C. elegans."

Tsukamoto, Satomi, Mori, Ikue, Nakano, Shunji

[International Worm Meeting, 2013]

Biogenic amines are important neurotransmitters or neuromodulators that regulate behavior in both vertebrates and invertebrates. To study the mechanisms by which biogenic amines regulate neural circuits, we are investigating thermotaxis behavior in C. elegans. After cultivation at a certain temperature with food, the animals migrate to that cultivation temperature, whereas they move randomly on the thermal gradient after cultivation without food. A simple neural circuit has been identified for this feeding state-dependent thermotactic behavior (Mori and Ohshima, 1995; Kuhara, Okumura et al., 2008). Also, we previously reported that exogenous serotonin and octopamine can mimic well-fed or food-deprived states, respectively (Mohri et al., 2005), which suggested that endogenous serotonin and octopamine can modulate thermotaxis. In the current study, we found that octopamine-deficient tbh-1 mutants stayed at the cultivation temperature even when they were starved, while serotonin-deficient bas-1 and tph-1 mutants dispersed from the cultivation temperature in fed condition. Thus, octopamine and serotonin are required for starvation and food signaling, respectively. To identify octopamine receptor required for thermotaxis, we evaluated thermotactic behavior of mutants for previously discovered octopamine receptor genes (ser-3, ser-6, octr-1, tyra-3) and found that only octr-1 mutants showed a phenotype similar to that of octopamine-deficient tbh-1 mutants. We so far showed that octopamine and serotonin are physiological modulators of thermotaxis and drive animals to the opposite behavioral states. We are currently investigating how these two amines regulate the neural circuit of thermotaxis. Our study will shed light onto a circuit-level mechanism for biogenic amine signaling in the brain.