Yu Hayashi et al. (2006) East Asia C. elegans Meeting "Analysis of the significance and mechanism of neurite elimination using C. elegans as a model"

Takaaki Hirotsu, Yuichi Iino, Yu Hayashi, Takeo Kubo, Hirofumi Kunitomo, Eriko Kage, Hideaki Takeuchi

[East Asia C. elegans Meeting, 2006]

During brain development, a large amount of neurites once formed undergo elimination, the mechanism and physiological significance of which are poorly understood. We previously showed that elimination of neurites occurs in the nematode C. elegans. In C. elegans, immediately after hatching, a pair of interneurons termed AIM are connected to each other by a short neurite, and this neurite is degenerated during the late L1 stage. We also showed that a highly conserved helix-turn-helix transcription factor MBR-1 is involved in this process. In the present study, in order to elucidate the molecular mechanism of neurite elimination, we aimed to identify components of the MBR-1 cascade. First, to examine how the expression of MBR-1 is regulated, we focused on the POU homeodomain transcription factor UNC-86 because its binding element was detected in the mbr-1 promoter region. As a result, in unc-86 mutants, the expression of mbr-1 promoter::gfp was attenuated in AIM neurons, indicating that UNC-86 promotes mbr-1 expression. Furthermore, in unc-86 mutants, the elimination of excessive AIM neurites was also defective, demonstrating that UNC-86 functions upstream of mbr-1 in neurite elimination. We are currently searching for factors directly involved in neurite elimination by comparing gene expression patterns between wild-type and mbr-1 mutants using the cDNA microarray method. In addition, we are also attempting to clarify the significance of neurite elimination. The mbr-1 mutant is defective in ""early adaptation"", a type of behavioral plasticity where chemotaxis towards attractive odorants is reduced after 5 min of pre-exposure to the same odorants, suggesting that MBR-1 is required for the development of the olfactory circuit. By executing cell-specific rescue experiments, we suggest that mbr-1 functions in a set of interneurons involved in olfactory processing. We expect that our research provides a useful model for genetic dissection of neurite elimination.

Yu Hayashi et al. (2004) East Asia Worm Meeting "MBR-1, a novel helix-turn-helix transcription factor, is involved in olfactory plasticity and elimination of excessive neurites in C. elegans"

Hideaki Takeuchi, Takaaki Hirotsu, Hirofumi Kunitomo, Takeo Kubo, Yuichi Iino, Yu Hayashi, Eriko Kage

[East Asia Worm Meeting, 2004]

Elimination of excessive neurites for the construction of functional neural circuits is commonly observed in the developing brain of various animals. The molecular basis of this phenomenon, however, is poorly understood. Here, we provide evidence that specific neurites of the simple model animal C. elegans also undergo elimination and that a novel helix-turn-helix transcription factor MBR-1 is involved in this process. We previously identified a novel transcription factor Mblk-1, which is expressed selectively in the honeybee brain. Subsequently, we identified its C. elegans homologue, MBR-1, and demonstrated its roles in "early adaptation", which is a novel type of behavioral plasticity where chemotaxis towards chemoattractive odorants is reduced after 5 min of pre-exposure to the same odorants. In the present study, we examined the possible roles of MBR-1 in neural circuit formation. Expression analysis using a translational reporter mbr-1::gfp revealed that MBR-1 is localized to the nuclei of various neurons in the head and tail. We then compared the morphology of the mbr-1 -expressing neurons between wild-type and mbr-1 mutant nematodes using a transcriptional reporter mbr-1 promoter::gfp . An extra pair of neurites, which connects the left and right AIM interneurons, was observed in the mbr-1 mutant adults, but not in the wild-type adults. This connection, however, was observed both in wild-type and mbr-1 mutant nematodes at the early larval stages. These results indicate that excessive neurites of the AIM interneurons are eliminated during the larval stages and that MBR-1 is involved in this process. Furthermore, we found that expression of zig-3 , which encodes a secretory protein with two immunoglobulin domains, is attenuated in the AIM interneurons in the mbr-1 mutants, suggesting that MBR-1 regulates the expression of zig-3 . Our results indicate that MBR-1 represents a novel class of transcription factors involved both in olfactory plasticity and the elimination of excessive neurites.

Yu Hayashi et al. (2005) International Worm Meeting "MBR-1, a novel helix-turn-helix transcription factor, is involved in olfactory plasticity and pruning of excessive neurites in C. elegans"

Hirofumi Kunitomo, Hideaki Takeuchi, Eriko Kage, Yu Hayashi, Takeo Kubo, Yuichi Iino, Takaaki Hirotsu

[International Worm Meeting, 2005]

In the developing brain, excessive neurites are actively pruned for the construction and remodeling of the neural circuit. The molecular basis of neurite pruning, however, is poorly understood. In this report, we show that the pruning of neurites also occurs in the simple neural circuit of Caenorhabditis elegans, and that a novel helix-turn-helix transcription factor MBR-1 is involved in this process. We previously identified Mblk-1, which is expressed selectively in the mushroom bodies (regions involved in memory formation and sensory integration) of the honeybee brain. Subsequently, we identified its C. elegans homologue, MBR-1, and demonstrated its roles in early adaptation, a novel type of behavioral plasticity in which chemotaxis towards a chemoattractive odorant becomes reduced after 5 minutes of pre-exposure to the same odorant. In the present study, we examined the possible roles of MBR-1 in neural circuit formation. Expression analysis using a translational reporter mbr-1::gfp revealed that MBR-1 is localized to the nuclei of various neurons in the head and tail ganglia. We then compared the morphology of mbr-1-expressing neurons between wild-type and mbr-1 mutant nematodes using a transcriptional reporter mbr-1 promoter::gfp. As a result, we found that excessive neurites connecting a pair of interneurons in the ventral ganglion, AIML/R, are eliminated during the larval stages in wild-type nematodes, but are frequently sustained till the adult stage in mbr-1 mutant nematodes. These results indicate that excessive neurites of the AIM interneurons are naturally pruned during the larval stages, and that MBR-1 is involved in this process. Furthermore, we show that mbr-1 expression in AIM neurons is regulated by the POU domain transcription factor UNC-86, while MBR-1 itself is involved in the regulation of the expression of zig-3, which encodes a neurosecretory protein. Our results demonstrate that MBR-1 represents a novel class of transcription factors involved both in olfactory plasticity and the pruning of neurites, and we also propose that C. elegans is a useful model for the analysis of the mechanisms of neurite pruning.

Eriko Kage et al. (2002) West Coast Worm Meeting "Functional analysis of the C.elegans homologue of Mblk-1, a transcription factor expressed in the honeybee brain"

Hirofumi Kunitomo, Hideaki Takeuchi, Masayuki Yamamoto, Takeo Kubo, Eriko Kage, Takaaki Hirotsu, Yuichi Iino, Asako Sugimoto, Takao Inoue, Hiroyuki Arai

[West Coast Worm Meeting, 2002]

Sensory processing is mediated by interneurons in various animal species. In insect, interneurons of the mushroom bodies (MBs) of the brain are responsible for sensory integration, memory and learning. We have previously identified a honeybee gene, termed Mblk-1, encoding a transcription factor that is expressed preferentially in the MB-interneurons of the honeybee brain. Homology search revealed that the DNA binding motif of Mblk-1 has significant sequence homology with those encoded by genes from various animal species, including the nematoda, fruit fly, mouse and human, suggesting that the functions of these proteins in neural system are conserved among these animals.

Yuichi Iino et al. (2003) International Worm Meeting "Interneuron-selective transcription factor MBR-1 is involved in olfactory plasticity"

Yu Hayashi, Takao Inoue, Takeo Kubo, Takaaki Hirotsu, Hideaki Takeuchi, Hiroyuki Arai, Hirofumi Kunitomo, Masayuki Yamamoto, Yoshihisa Mita, Yuichi Iino

[International Worm Meeting, 2003]

Processing of sensory information is mediated by specialized interneurons in various sensory systems. In insects, interneurons in the mushroom body of the brain (MB interneurons) are important for sensory integration and olfactory learning. We have previously identified the Mblk-1 gene, which encodes a novel transcription factor that is expressed preferentially in the MB interneurons of the honeybee brain.Homology searches revealed that the DNA binding motifs of Mblk-1 have significant sequence similarities with those encoded by a set of genes from nematode, fruit fly, mouse and human. However, the function of these proteins in the nervous system remains unclear. To clarify the role of Mblk-1 homologues, we performed a reverse genetic analysis using C. elegans. We identified a gene, termed mbr-1 (Mblk-1 related factor-1), which carries two DNA binding motifs with significant homologies with those of Mblk-1. mbr-1 promoter::GFP is expressed selectively in restricted sets of head interneurons identified as AIM, RIC, etc. GFP expression is first observed around the 3-fold stage and continues throughout the larval and adult stages, suggesting the possibility that mbr-1 plays a role in differentiation and/or maintenance of the function of these neurons.To investigate mbr-1 functions in sensory processing, an mbr-1 deletion mutant was isolated by the UV/TMP method and tested in chemotaxis and olfactory adaptation assays. Wild-type worms show reduced chemotaxis towards attractive odorants (benzaldehyde, isoamylalcohol and pyrazine) after pre-exposure to the same odorants for 5 min (Hirotsu and Iino, 2002 West Coast Worm Meeting). In contrast, the mbr-1 mutant worms showed a defect in the adaptation to benzaldehyde, while they showed normal plasticity with isoamylalcohol and pyrazine. The defect was rescued by a wild-type mbr-1 transgene, indicating that mbr-1 is involved in adaptation to benzaldehyde. These findings suggest that the transcription factor MBR-1 acts in specific interneuron(s) for olfactory adaptation. We are attempting cell type-specific rescues to identify the interneuron(s) where mbr-1 functions.

Karla Opperman et al. (2007) International Worm Meeting "A structure-function analysis of the C. elegans L1CAMs."

Lihsia Chen, Xuelin Wang, Karla Opperman, Shan Zhou

[International Worm Meeting, 2007]

Cell adhesion molecules play important roles during development and in maintaining tissue integrity. L1CAMs are a family of immunoglobulin (Ig)-like cell adhesion molecules that are important in vertebrate nervous system development. L1CAMs are conserved in C. elegans, and are encoded by the lad-1/sax-7 and lad-2 genes. SAX-7 and LAD-2 have distinct functions in the nervous system despite overlapping neuronal expression. SAX-7 is required to maintain neuronal positioning while LAD-2 participates in axon pathfinding. We are performing a structure-function analysis to determine how both proteins mediate their functions. Because the cytoplasmic tails between both proteins are distinct, we are focusing our analysis on how the cytoplasmic tail regulates L1CAM functions. Indeed, while the extracellular domains of SAX-7 and LAD-2 are conserved, the LAD-2 cytoplasmic tail is completely divergent and does not contain any of motifs conserved in SAX-7 and vertebrate L1CAMs. These motifs include the ankyrin-binding motif, which links L1CAMs to the spectrin-actin cytoskeleton, the PDZ-binding motif, and conserved tyrosine residues that are phosphorylated. Our preliminary results reveal that both conserved motifs, as well as phosphorylation one of the tyrosine residues, which regulates ankyrin binding, all contribute to the function of SAX-7 in neuronal position maintenance. (1) Chen L, Ong B, Bennett V. J Cell Biol. 2001 Aug 20;154(4):841-55. (2) Wang X, Kweon J, Larson S, Chen L. Dev Biol. 2005 Aug 15;284(2):273-91. (3) Sasakura H, Inada H, Kuhara A, Fusaoka E, Takemoto D, Takeuchi K, Mori I. EMBO J. 2005 Apr 6;24(7):1477-88. Epub 2005 Mar 17.