Yuji Kohara (1999) International C. elegans Meeting "Post genomics strategies and resources in C.elegans"

Yuji KOHARA

[International C. elegans Meeting, 1999]

We will present our plans for genome biology of the worm based on our cDNA project and wish to discuss about possible coordination in the worm community. 1) More cDNA clones and full-length libraries: Thus far, we have analyzed some 65,000 cDNA clones and classified them into about 10,000 unique cDNA groups. Since the consortium has made the prediction of most of genes, our effort has been shifted to expression and function analysis rather than extension of the EST analysis. However, our recent analysis indicated that EST information is still essential to determine the gene structures (see Thierry-Mieg et al.). Thus, we are planning to continue the EST analysis together with full-sequencing of the clones that showed alternative splicing. In parallel, we plan to construct full-length cDNA libraries using the oligo capping method developed by Sugano's group at the University of Tokyo. 2) Expression analysis by in situ hybridization: We have done in situ hybridization with 3,500 gene probes mostly from LG3 and LGX, and will finish the remaining 7,000 gene within a year. However, since giving of full annotation of the expression patterns is a time consuming task, we intend to release typical images and their brief description at our NEXTDB WWW site before adding full annotation. 3) Promoters for specific transcription: The in situ analysis has identified a lot of genes that show specific mRNA distribution. The promoters of these genes should be very useful for ectopic gene expression, cell/lineage/stage specific RNAi and so on. Therefore, we plan to identify such promoter fragments systematically. 4) Microarray and systematic RNAi: We selected some 8,000 representative clones (1-2Kb insert clones suitable for PCR) and are producing microarrays on glass slides (version 1). Preparation of the remaining 2,000 representative clones is also in progress to add the microarray (version 2). The same set could be applied to systematic RNAi. We are preparing to make these resources available to the community. 5) Others: Antibodies, deletion mutant bank (in collaboration with Shohei Mitani at Tokyo Women's Medical College), computer simulation of development, and so on.

Yuji Kohara et al. (2001) International C. elegans Meeting "Developmental expression map of C.elegans genome"

Ikuko Sugiura, Masahiro Ito, Keiko Hirono, Yuji KOHARA, Michiko Serizawa, Tokie Oba, Takami Suzuki, Yohei Minakuchi, Kyoko Nakata, Masumi Obara, Mina Iwata, Tadasu SHIN-I, Yumiko Ueta

[International C. elegans Meeting, 2001]

We have been performing whole mount in situ hybridization using the cDNAs that have been classified in our EST project (See Thierry-Mieg et al.) as gene probes. Although there was a big delay in the project last year because we needed almost half a year to fix problems due to which our standard protocol suddenly didn't work, now we get the project back on the track and have finished some 7,600 genes. We have given minimal annotation to the in situ results; 10 stages for embryogenesis, 4 stages for larval-adult stage, on average 10 patterns (cell(s), tissue, region) per stage, 3 levels of relative intensity of signals per each pattern. Using the information, clustering analysis and extraction of consensus sequences in the upstream regulatory regions are in progress (see Ito et al.). Subsets of genes whose expression patterns are identical are being subjected to various analysis including RNAi, immunostaining and bioinformatic analysis (motif search, for example). This is a unique and powerful approach and, for example, we identified the function of proteasome in oocyte maturation and fertilization (see Hirono et al.). All the relevant data are integrated in NEXTDB and we are preparing to make them open by the worm meeting (See the demo by Shin-i et al.). In this report, we will show the content and the results of studies based on the database. Furthermore, we are planning "Annotation Relay" in which experts in various fields are invited to this lab by turns (a week or two per round and several people per round depending on the number of microscope) and look at the in situ slides on the microscope with the expert eyes to give more precise and deeper annotations to the in situ data than those we gave.

Kagoshima, Hiroshi et al. (2011) International Worm Meeting "The homeobox transcription factors, CEH-14 and TTX-1, regulate expression of gcy-8 and gcy-18 in C. elegans."

Kagoshima, Hiroshi, Kohara, Yuji

[International Worm Meeting, 2011]

To investigate mechanism of cell-specific transcription, we chose AFD thermosensory neuron for our target cell and have analyzed nine AFD-specific promoters. Among these promoters, we focused on analysis of gcy-8 and gcy-18 promoters which have arisen from recent gene duplication, suggesting they may conserve upstream regulatory mechanisms. Indeed, reporter expressions from both promoters were downregulated in either mutant of the homeobox genes, ttx-1 or ceh-14, and the expressions of gcy-8 and gcy-18 were completely abolished under ttx-1; ceh-14 double mutant background. Moreover, forced expression of both the transcription factors in AWB chemosensory neuron induced ectopic expression of gcy-18 in AWB. These results suggest that ttx-1 and ceh-14 transcription factors play pivotal roles in gcy-8 and gcy-18 expressions. We also showed direct interaction of TTX-1 and CEH-14 proteins with gcy-8 and gcy-18 promoters by gel shift assay using recombinant GST fusion proteins, and identified the binding sites of the transcription factors. We are currently analyzing mutated promoter constructs to confirm if these sites determined by in vitro analysis are required for in vivo function.

Kagoshima, Hiroshi et al. (2013) International Worm Meeting "The homeobox transcription factors, CEH-14 and TTX-1 regulate the AFD neuron specific gene expression of gcy-8 and gcy-18 in C. elegans."

Kohara, Yuji, Kagoshima, Hiroshi

[International Worm Meeting, 2013]

The identities of a wide variety of cells in multicellular organisms are given by the expression of characteristic sets of genes, mainly controlled by cell-specific transcriptional regulation. In particular, to generate different types of neurons, combinatorial expression of multiple transcription factors play pivotal roles for cell-specific gene expression. To understand the mechanism of the cell-specific transcriptional regulation, we used a pair of AFD thermosensory neurons in C. elegans as a model system, and analyzed the regulation of guanylyl cyclase genes, gcy-8 and gcy-18, exclusively expressed in AFD. Here we show that the AFD-specific expression of gcy-8 and gcy-18 requires the simultaneous expression of homeodomain proteins, CEH-14/LHX3 and TTX-1/OTX1 in AFD neuron. We found the expression of gcy-8 and gcy-18 in AFD was downregulated by either mutation of ttx-1 or ceh-14, and completely lost in the double mutants. We showed direct interaction of CEH-14 and TTX-1 proteins with gcy-8 and gcy-18 promoters by EMSA, and identified the binding sites of CEH-14 and TTX-1. Transgenic strains carrying gcy-8 and gcy-18 reporters with CEH-14 and/or TTX-1 sites mutation revealed that those binding sites were essential for AFD-specific expression of gcy-8 and gcy-18. We also demonstrated that simultaneous forced expression of CEH-14 and TTX-1 in AWB chemosensory neuron could induce ectopic expression of gcy-8::GFP and gcy-18::GFP in AWB. Finally we showed that the regulation of gcy-8 and gcy-18 expression by ceh-14 and ttx-1 is evolutionally conserved in five Caenorhabditis species. Although thermosensory- and chemosensory-neurons have different functions, those sensory neurons may share potential exchangeability, and ceh-14 and ttx-1 confer AFD identity at the final step of the cell specification.

SJ Kwon et al. (1999) International C. elegans Meeting "Characterization of a C. elegans homologue of band 4.1 protein"

SJ Kwon, JH Ahnn, SK Jung

[International C. elegans Meeting, 1999]

Band 4.1 is a cytoskeleton protein found in human erythrocyte. It has been reported that Band 4.1 regulates membrane mechanical properties through modulation of interaction between spectrin and actin, and also regulates band 3-ankyrin interaction. However, the exact biochemical and pyshiological function of this protein has not been elucidated. When we searched the Yuji Kohara 's in situ database for genes specially expressed in body-wall muscle, we came across a clone, yk21h2 . This clone appeared to be expressed only in body-wall muscle during embryogenesis. We found a gene, F43d9.1 , which shows high homology with band 4.1 protein. This protein is highly conserved in the other organisms. It has 83% identity with band 4.1 protein. We are interested in the facts that C. elegans does not have erythrocytes but this protein is so highly conserved that we can suggest it in an evolutional aspect. According to In situ data( Yuji Kohara ), it is transcripted along the body-wall muscle during embryogenesis. So we expect that this protein may be associated with muscle development. Now we are trying to characterize it in C. elegans . To investigate the pyshiological role of this protein, we are trying to see the expression pattern in the C. elegans , and to get the deletion mutant using EMS mutagenesis.

Andachi, Yoshiki et al. (2011) International Worm Meeting "In situ hybridization analysis of let-7 microRNA."

Andachi, Yoshiki, Kohara, Yuji

[International Worm Meeting, 2011]

MicroRNAs (miRNAs) are short RNAs that down-regulate target genes by forming base pairs with their mRNAs. miRNA genes are transcribed into long transcripts, primary miRNAs (pri-miRNAs), which are subsequently cleaved into mature miRNAs. Though miRNA expression is under transcriptional control, several miRNAs are also post-transcriptionally regulated. One of the miRNAs is let-7 in C. elegans, which is temporally regulated. It remains unknown whether let-7 is also post-transcriptionally regulated in a tissue-specific manner because of the unavailability of a method for in situ detection of miRNA in C. elegans. We previously developed a strategy for isolating cDNA clones of target mRNAs that form base pairs with a miRNA of interest, in which the cDNAs are synthesized from the mRNAs using the miRNA as a reverse-transcription primer. Application of the method to let-7 family miRNAs, mir-48 and mir-84, provided clones derived from parts of the let-7 pri-miRNA. In a region 3' adjacent to the clones, three sequences complementary to the seed of the let-7 family miRNAs are included, and the seed complementary sequences are conserved in corresponding regions of the let-7 genes in C. briggsae, C. remanei and C. brenneri. One possibility deduced from the findings is that the let-7 family miRNAs are involved in let-7 biogenesis by binding to the seed complementary sequences. To examine tissues where let-7 miRNA is produced, we developed an in situ hybridization (ISH) method, based on the ISH method to know mRNA expression in our lab. Using the ISH method, let-7 miRNA was detected in vulva, rectum and pharynx of N2 worms at the L4 stage. ISH analysis of let-7(mn112) deletion mutants rescued by extra-chromosomal arrays of a let-7 gene clone exhibited intense signals in seam, ventral neurons and distal tip cells in addition to the above tissues, presumably due to increased let-7 gene copy number. As for mir-48, miRNA expression was observed in vulva, rectum, seam and a few neurons around pharynx of N2 worms, indicating that both let-7 and mir-48 miRNAs were co-expressed in several tissues. To test the requirement of the seed complementary sequences of the let-7 pri-miRNA for let-7 biogenesis, a let-7 gene clone with all the complementary sequences substituted was prepared and introduced into let-7(mn112) mutants. Extra-chromosomal arrays of the clone turned out to rescue the lethal phenotype of let-7 mutants, and ISH analysis of the transgenic strains provided essentially the same let-7 expression pattern as the transgenic strains of the wild-type clone. Further substitution studies are in progress.

Andachi, Yoshiki et al. (2013) International Worm Meeting "LIN-28-dependent repression of let-7 miRNA is required for oogenesis."

Kohara, Yuji, Andachi, Yoshiki

[International Worm Meeting, 2013]

let-7 microRNA (miRNA) plays important roles in various phases of development. The maturation of let-7 miRNA is inhibited by binding of LIN-28 protein to precursors of the miRNA at early larval stages. Although lin-28 mutants show a precocious phenotype at the L2 stage in seam cells and a protruding vulva phenotype, the defects are not suppressed by let-7 mutation. To elucidate the biological significance of LIN-28-dependent repression of let-7 miRNA, we examined the gonad because some lin-28 mutant worms were sterile. While the ventral half of the adult gonad consists of a row of oocytes in wild-type worms, the region occupied by oocytes was shortened proximally and the syncytium containing pachytene-stage nuclei extended to the remaining part in lin-28 mutants. The gonadal abnormalities was not only rescued by lin-28 transgenes, but also suppressed by let-7 mutation. In the syncytium adjacent to oocytes, MPK-1 is activated by phosphorylation in wild-type worms. The activated MPK-1 was reduced in lin-28 mutants, but not in lin-28; let-7 double mutants. We searched for target genes of the ectopically expressed let-7 miRNAs by our screening method, in which endogenous miRNAs binding to mRNAs were used as reverse-transcription primers for the synthesis of cDNAs from the mRNAs, and clones of the cDNAs were sequenced to identify target genes. Several genes were obtained both from lin-28 mutant worms and lin-28(RNAi) worms, but not from worms RNAi-depleted for control. Of them, gonadal defects similar to but weaker than those in lin-28 mutants were observed in pqn-47 mutants whose L1 arrest phenotype was rescued by an extra-chromosomal array including the pqn-47 coding sequence. Triple mutant analysis revealed that pqn-47 was epistatic to lin-28; let-7. hbl-1 mutants also exhibited the extension of the syncytium to the ventral side at low penetrance, and the mutation enhanced the gonadal defects in pqn-47 mutants. The activated MPK-1 was reduced in pqn-47; hbl-1 double mutants. These results suggest that LIN-28-dependent repression of let-7 miRNA is required for oogenesis, and the gonadal defects in lin-28 mutants are due to down-regulation of both pqn-47 and hbl-1 by ectopically expressed let-7 miRNAs.

Kagoshima, Hiroshi et al. (2011) International Worm Meeting "Functional genomics of two Antarctic nematodes, Panagrolaimus davidi and Plectus murrayi."

Kohara, Yuji, Kagoshima, Hiroshi

[International Worm Meeting, 2011]

Antarctica is an extreme environment for life. Antarctic animals must remain either frozen or dry for much of the year, and they can grow only during short period when liquid water is available from melting snow in the summer. Even then, they are exposed to periodical freeze-thaw. We aim to elucidate how the Antarctic animals adapt to the extreme environment, using two Antarctic nematodes. (1) Panagrolaimus davidi: We analyzed 25,000 cDNAs prepared from P. davidi cultivated at 20 deg C with water and food (good condition for therir growth). We found P. davidi constitutively expressed high level of stress response genes, such as heat resistant gene, lea-1, even in the good condition. We are currently performing sequence analysis of another cDNA library generated from P. davidi cultivated at 4 deg C (cold-stressed condition), to screen candidate genes for cold tolerance, by the comparison of expression profiles between two libraries. (2) Plectus murrayi: We were able to recover nematodes from a frozen moss sample collected 25.5 years ago, near Showa station, eastern Antarctica. We identified this nematodes as Plectus murrayi using morphological keys and confirmed it by their 18S and 28S rRNA sequences. They can grow on water agar plates and feed bacteria grew from the original isolates (most probably, Pseudomonas sp. derived from Antarctica). They tolerated freezing stress (-20 deg C and -80 deg C for 24 hrs) and desiccation stress (dried at 98% and 76.5% relative humidity for 24 hrs).

Noguchi, Kouki et al. (2009) International Worm Meeting "Studies on localization mechanisms of the maternal pos-1 mRNA in C. elegans embryos."

Kohara, Yuji, Noguchi, Kouki

[International Worm Meeting, 2009]

mRNA localization has been observed in diverse cells and organisms. In C. elegans, there are also several maternal mRNAs, which is known to be localized during early development. Distribution patterns of these mRNAs suggest involvement of mRNA degradation in the localization. However, there is little information about cis- and trans-elements for the mRNA localization. To understand how cells regulate the localization, identification of these elements are desired. In this study, we are focusing on the maternal pos-1 mRNA. The pos-1 mRNA is localized to the posterior half of embryos during the 1st cleavage and is localized to germ lineage in early development. To investigate cis-elements of the localization, we first developed a vector, which maternally expresses fusion mRNA of VENUS CDS and arbitrary sequences from the pos-1 promoter. By using the vector, we generated transgenic worms that express the VENUS::pos-1 3''UTR fusion mRNA and tested localization of the mRNA. In situ hybridization revealed that the mRNA is localized as intrinsic pos-1 mRNA. Thus, we analyzed the pos-1 sequences by using the vector. Deletion analysis of the pos-1 3''UTR revealed that 59 nt of the pos-1 3''UTR (bases 178-236 downstream of the pos-1 stop codon.) is required for the localization. Interestingly, there are evolutionary conserved CYCACA tandem repeats (191-196 and 199-204) and 30nt (207-236). Hence, we next tested whether these sequences are involved in the localization by making series of constructs, which contain substitutions in these sequences. This analysis revealed that these conserved sequences are actually required for the localization. We also found that, the VENUS protein is also localized in the transgenic embryos of VENUS::pos-1 3''UTR mRNA. In the embryos, the VENUS protein was first detected just after fertilization and was uniformly expressed at the time. After that, the protein was preferentially accumulated in posterior half by the four-cell stage. Such localization is not observed in the VENUS::mel-11 3''UTR worms; in these worms, the VENUS protein was expressed in adult gonad and was uniformly distributed in early embryos. Thus, the VENUS protein localization is pos-1 3''UTR dependent and should be accomplished by local translation rather than post-translational protein localization. Strikingly, the pos-1 3''UTR dependent protein localization is abolished in mex-5 mutant, in which the pos-1 mRNA localization is also abolished. It suggests a close correlation between pos-1 3''UTR dependent local translation and mRNA localization. We are now trying to identify trans-elements of the pos-1 mRNA localization by using yeast three-hybrid assay. At the meeting, we will also presents the result.

Kagoshima, Hiroshi et al. (2013) International Worm Meeting "LEA genes in Antarctic nematode, Panagrolaimus davidi."

Kohara, Yuji, Kagoshima, Hiroshi

[International Worm Meeting, 2013]

In Antarctica, terrestrial animals live in one of the most extreme environments on Earth. They normally can be active and grow only during the brief summer when sufficient heat is available in their microhabitats for liquid water to be present from melting snow or other sources. However, they have to withstand freezing and desiccation stresses for long periods over winter, and periodically (and often unpredictably) at other times of the year. We would like to know how the Antarctic animals adapt to the extreme environment, using Antarctic nematode Panagrolaimus davidi as a model organism. We analyzed total 50,000 ESTs prepared from P. davidi cultivated at 20 deg C (good condition for growth) and at 4 deg C (cold-stressed condition). We found several candidate genes for environmental tolerances in the cDNA library. In particular, we picked up LEA genes for further analysis. The LEA gene (named after "Late Embryo Abundant") was originally found in wheat seed. This gene has been isolated from plants, bacteria and animals, which have environmental tolerances. The LEA genes share characteristic 11 amino acids repeat (LEA repeat), which adopt amphipathic α-helix during desiccation condition, and may function in retention of water and protection of proteins/membrane. P. davidi had more than 10 LEA genes and some of them had highly diverged LEA repeats, suggesting they may have different functions from that of typical LEA genes. We are analyzing P. davidi LEA genes in vitro by recombinant LEA proteins and in vivo by transgenic C. elegans carrying P. davidi LEA genes.