{"created":"2023-06-20T13:20:51.589696+00:00","id":921,"links":{},"metadata":{"_buckets":{"deposit":"4f11492c-5269-4398-9f94-d62d70bafe2e"},"_deposit":{"created_by":1,"id":"921","owners":[1],"pid":{"revision_id":0,"type":"depid","value":"921"},"status":"published"},"_oai":{"id":"oai:ir.soken.ac.jp:00000921","sets":["2:430:20"]},"author_link":["9918","9920","9919"],"item_1_creator_2":{"attribute_name":"著者名","attribute_type":"creator","attribute_value_mlt":[{"creatorNames":[{"creatorName":"鈴木, 教郎"}],"nameIdentifiers":[{}]}]},"item_1_creator_3":{"attribute_name":"フリガナ","attribute_type":"creator","attribute_value_mlt":[{"creatorNames":[{"creatorName":"スズキ, ノリオ"}],"nameIdentifiers":[{}]}]},"item_1_date_granted_11":{"attribute_name":"学位授与年月日","attribute_value_mlt":[{"subitem_dategranted":"1997-03-24"}]},"item_1_degree_grantor_5":{"attribute_name":"学位授与機関","attribute_value_mlt":[{"subitem_degreegrantor":[{"subitem_degreegrantor_name":"総合研究大学院大学"}]}]},"item_1_degree_name_6":{"attribute_name":"学位名","attribute_value_mlt":[{"subitem_degreename":"博士（理学）"}]},"item_1_description_12":{"attribute_name":"要旨","attribute_value_mlt":[{"subitem_description":" The nematode Caenorhabditis elegans (abbreviated as C.elegans below) has several advantages as a model organism for studying the nervous system. First, its nervous system is very simple. C.elegans has 959 cells, of which 302 are neurons that constitute the nervous system. Second, the complete structure of the nervous system, the identity of each neuron and the connections by chemical synapses and gap junctions, has been determined by electron microscopic analysis of the serial sections of worm body. Third, genetic methods are available. Mutants can be isolated easily after mutagenesis of self-fertilizing hermaphrodites. Furthermore, males are produced spontaneously among the progeny of hermaphrodites at a low frequency by X chromosome non-disjunction. Males can mate with hermaphrodites and therefore can be used for mapping mutations. Fourth, it is possible to kill specific neurons by laser ablation under a light microscope. This technique is useful in determining the role of each type of neuron.
The functions of the nervous system can be studied genetically by isolating and characterizing mutants that are abnormal in various behaviors. Mutants of C.elegans that are abnormal in locomotion, egg-laying, pharyngeal pumping, chemotaxis, defecation etc. have been isolated and characterized in the past over 20 years. However, in C.elegans, functions of the nervous system can be studied also by a specific aspect of post-embryonic development, i.e., regulation of dauer larva formation.
The dauer larva is a special third-stage larva produced under harsh conditions. The life cycle of C.elegans takes three and a half days at 20 'C. After hatching, C.elegans usually passes through 4 larval stages called L1, L2, L3 and L4, before becoming an adult worm. However, if there are little food and much pheromone (due to overcrowding) around the worm, it becomes a dauer larva instead of an L3 larva. Temperature is another factor that modifies the decision between dauer and L3. At a high temperature more dauer larvae tend to be formed than at a low temperature. The dauer larva has a characteristic dark, thin body. It does not feed, because its mouth is closed. It can survive up to three months, and when it encounters food, it molts and goes into the L4 larval stage.
The two main environmental cues for the dauer/L3 decision, namely food and pheromone, seem to be sensed by the nervous system. C.elegans has a pair of sensory organs called amphids in the head. It is known that if we kill two types of sensory neurons (ADF and ASI) in the amphids of wild type worms, they become dauer larvae at a certain probability even under non-dauer-forming conditions. The probability increases to nearly 100%, if we also kill another type of amphid sensory neurons (ASG).
Many mutants that are abnormal in the dauer larva formation have been isolated and named daf mutants. They are classified into two groups. One is called dauer-constitutive (daf-c) mutants, which form dauer larvae even under non-dauer-forming conditions, i.e., much food and low pheromone concentration. The other is called dauer-defective (daf-d) mutants, which do not form dauer larvae even under dauer-forming conditions, i.e., little food and high pheromone concentration. Some of the daf-d mutants have defects in the structure of amphids. According to the electron microscopic structure and the absence of uptake of some fluorescent dyes by amphids and phasmids, those defects seem to prevent the outer cues from getting access to the amphid chemosensory neurons. This is another piece of evidence showing that dauer formation is controlled by the nervous system.
Although about 30 daf genes and about 20 other genes are known to affect dauer formation, the number is still much less than the expected number of genes that affect neurons. Part of the reasons may be related to the results of the cell-killing experiments mentioned above. Since at least three types of sensory neurons act in parallel in the regulation of dauer formation, the number of neural mutations that cause defects in all of them may be limited. This implies that there may be many neural mutations that show the synthetic Daf phenotype, namely, exhibit abnormality in dauer formation only if two or more of them are combined to form double- or multiple-mutants. Two examples of the synthetic dauer-constitutive (Sdf-c) phenotype were known when I started this study: unc-31 ;unc-3 and unc-31 ;aex-3. In these cases the Sdf-c phenotype was found somewhat accidentally, while each of the mutations was isolated by abnormalities in locomotion (in the cases of unc-3 and unc-31) or in defecation (in the case of aex-3).
I thought it would be possible to identify many new neural genes by isolating new sdf-c mutants on the unc-31 background. I planned to use unc-31 as the first mutation mainly for two reasons. First, both of the two known Sdf-c pairs had unc-31 as one of the mutations. Hence, it was expected that I would obtain unc-3 and aex-3 among the new mutations, if I used unc-31 as the first mutation. This would serve as a positive control showing that the procedure of the mutant isolation was correct. Second, it was known that the unc-31 mutant forms dauer larvae under non-dauer-forming conditions, if only the ASI sensory neurons are killed, while killing of both ASH and ADF was necessary to make wild-type worms dauer-constitutive. This means that many, if not all, of
the sdf-c mutants isolated on the unc-31 background have defects in the ASH neuron. Mutants that have defects in a specific type of neurons will be useful in the future analysis of the nervous system by genetic means.
Thus, I isolated mutants that exhibit the Sdf-c phenotype on unc-31 background. For this purpose I used unc-31(e169); utEx[unc-31(+)], an unc-31 strain in which a clone of the wild-type unc-31 gene exists as an extrachromosomal array. Since the extrachromosomal array is lost at a low frequency, this strain segregates both Unc+ and Unc worms. The former have the parental genotype, unc-31(e169); utEx[unc-31(+)], and the latter, unc-31(e169). I mutagenized unc-31(e169); Ex[unc-31(+)] with ethyl methanesulfonate and screened 5539 of F1 Unc+ progeny for those which segregate Unc dauer larvae but not Unc+ dauer larvae under non-dauer-forming conditions. Those which segregate both Unc and Unc+ dauer larvae were discarded, because they were daf-c rather than sdf-c mutants. In this way I obtained 44 mutants that showed the Sdf-c phenotype on the unc-31 background.
I mapped 42 of the 44 mutants by using STS (Sequence-Tagged Site) markers and known mutations, while two of them had too low penetrance to be mapped. Eight of the mapped mutations were found to be alleies of four known genes by complementation tests (one allele of tax-2, two alleles of che-11, two alleles of osm-6 and three alleies of aex-3). The rest 34 mutations, which were most probably alleles of new genes, were classified into 18 complementation groups.
In the meantime, it was found in our laboratory that known daf-d mutations that are abnormal in dye-filling (Dyf phenotype) into amphids and phasmids have the Sdf-c phenotype if they were combined with the unc-31(el69) mutation. Therefore, I tested the 44 new mutations for the Dyf phenotype. Besides the each two alleles of che-11 and osm-6 two mutations in two new genes (Sdf-3(ut160) and sdf-13(utl82)) had the Dyf phenotype. In these mutants, environmental cues cannot reach the ASI neurons due to the structural abnormalities of amphids. In other mutants they probably can reach ASI but cannot be sensed by ASI, or the transmission of the signal may be blocked in ASI or downstream neurons.
Since the new mutations may cause abnormalities in neurons other than ASI, I checked some behaviors to detect such abnormalities. Some of the mutants were abnormal in osmotic avoidance (defect in ASI), while others were abnormal in chemotaxis to benzaldehyde (defect in AWC) or to diacetyl (defect in AWA). Especially interesting was sdf-1(ut161), which avoids benzaldehyde at a concentration that attracts wild-type worms.
In conclusion, this study shows that by isolating synthetic dauer-constitutive
mutations, we can identify many new neural genes that probably control reception, signal transmission, and signal processing of the environmental cues (pheromone and food) for dauer formation. The mutants obtained in this study will be useful for analyzing functions of ASI neurons in the future.","subitem_description_type":"Other"}]},"item_1_description_18":{"attribute_name":"フォーマット","attribute_value_mlt":[{"subitem_description":"application/pdf","subitem_description_type":"Other"}]},"item_1_description_7":{"attribute_name":"学位記番号","attribute_value_mlt":[{"subitem_description":"総研大甲第271号","subitem_description_type":"Other"}]},"item_1_select_14":{"attribute_name":"所蔵","attribute_value_mlt":[{"subitem_select_item":"有"}]},"item_1_select_8":{"attribute_name":"研究科","attribute_value_mlt":[{"subitem_select_item":"生命科学研究科"}]},"item_1_select_9":{"attribute_name":"専攻","attribute_value_mlt":[{"subitem_select_item":"18 遺伝学専攻"}]},"item_1_text_10":{"attribute_name":"学位授与年度","attribute_value_mlt":[{"subitem_text_value":"1996"}]},"item_creator":{"attribute_name":"著者","attribute_type":"creator","attribute_value_mlt":[{"creatorNames":[{"creatorName":"SUZUKI, Norio","creatorNameLang":"en"}],"nameIdentifiers":[{}]}]},"item_files":{"attribute_name":"ファイル情報","attribute_type":"file","attribute_value_mlt":[{"accessrole":"open_date","date":[{"dateType":"Available","dateValue":"2016-02-17"}],"displaytype":"simple","filename":"甲271_要旨.pdf","filesize":[{"value":"281.5 kB"}],"format":"application/pdf","licensetype":"license_11","mimetype":"application/pdf","url":{"label":"要旨・審査要旨 / Abstract, Screening Result","url":"https://ir.soken.ac.jp/record/921/files/甲271_要旨.pdf"},"version_id":"82923477-2822-43c9-bc3b-26520403ece6"},{"accessrole":"open_date","date":[{"dateType":"Available","dateValue":"2016-02-17"}],"displaytype":"simple","filename":"甲271_本文.pdf","filesize":[{"value":"6.9 MB"}],"format":"application/pdf","licensetype":"license_11","mimetype":"application/pdf","url":{"label":"本文","url":"https://ir.soken.ac.jp/record/921/files/甲271_本文.pdf"},"version_id":"1483063d-401f-477d-8ddb-ff61935880de"}]},"item_language":{"attribute_name":"言語","attribute_value_mlt":[{"subitem_language":"jpn"}]},"item_resource_type":{"attribute_name":"資源タイプ","attribute_value_mlt":[{"resourcetype":"thesis","resourceuri":"http://purl.org/coar/resource_type/c_46ec"}]},"item_title":"合成dauer構成性表現型を指標としたC.elegansの神経系変異の分離と解析","item_titles":{"attribute_name":"タイトル","attribute_value_mlt":[{"subitem_title":"合成dauer構成性表現型を指標としたC.elegansの神経系変異の分離と解析"},{"subitem_title":"Isolation and Analysis of Synthetic Dauer- Constitutive Mutants Defective in the NervousSystem in C.elegans","subitem_title_language":"en"}]},"item_type_id":"1","owner":"1","path":["20"],"pubdate":{"attribute_name":"公開日","attribute_value":"2010-02-22"},"publish_date":"2010-02-22","publish_status":"0","recid":"921","relation_version_is_last":true,"title":["合成dauer構成性表現型を指標としたC.elegansの神経系変異の分離と解析"],"weko_creator_id":"1","weko_shared_id":1},"updated":"2023-06-20T14:42:28.914986+00:00"}