Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
Zoological Lett
2018 Jan 01;4:24. doi: 10.1186/s40851-018-0102-2.
Show Gene links
Show Anatomy links
Draft genome of Dugesia japonica provides insights into conserved regulatory elements of the brain restriction gene nou-darake in planarians.
An Y, Kawaguchi A, Zhao C, Toyoda A, Sharifi-Zarchi A, Mousavi SA, Bagherzadeh R, Inoue T, Ogino H, Fujiyama A, Chitsaz H, Baharvand H, Agata K.
???displayArticle.abstract???
BACKGROUND: Planarians are non-parasitic Platyhelminthes (flatworms) famous for their regeneration ability and for having a well-organized brain. Dugesia japonica is a typical planarian species that is widely distributed in the East Asia. Extensive cellular and molecular experimental methods have been developed to identify the functions of thousands of genes in this species, making this planarian a good experimental model for regeneration biology and neurobiology. However, no genome-level information is available for D. japonica, and few gene regulatory networks have been identified thus far.
RESULTS: To obtain whole-genome information on this species and to study its gene regulatory networks, we extracted genomic DNA from 200 planarians derived from a laboratory-bred asexual clonal strain, and sequenced 476 Gb of data by second-generation sequencing. Kmer frequency graphing and fosmid sequence analysis indicated a complex genome that would be difficult to assemble using second-generation sequencing short reads. To address this challenge, we developed a new assembly strategy and improved the de novo genome assembly, producing a 1.56 Gb genome sequence (DjGenome ver1.0, including 202,925 scaffolds and N50 length 27,741 bp) that covers 99.4% of all 19,543 genes in the assembled transcriptome, although the genome is fragmented as 80% of the genome consists of repeated sequences (genomic frequency ≥ 2). By genome comparison between two planarian genera, we identified conserved non-coding elements (CNEs), which are indicative of gene regulatory elements. Transgenic experiments using Xenopus laevis indicated that one of the CNEs in the Djndk gene may be a regulatory element, suggesting that the regulation of the ndk gene and the brain formation mechanism may be conserved between vertebrates and invertebrates.
CONCLUSION: This draft genome and CNE analysis will contribute to resolving gene regulatory networks in planarians. The genome database is available at: http://www.planarian.jp.
Figure 7. Transgenic experiments suggested that Djndk CNE3 might be a regulatory element. a The arrowhead shows CNE3 inserted actGFP vector, CNE (140)-actGFP driven GFP to express in the anterior region at the end of gastrulation in transgenic Xenopus embryos (n = 132). b Putative transcription factor-binding motifs are boxed in different colors; those subjected to mutation analysis are indicated by asterisks. The detailed point mutation design of three transcription factor-binding motifs are shown by red colored words. c Mutation analysis of CNE3 (the 140 bp element). actGFP is an empty reporter construct that contains the β-actin basal promoter; wt (140) is the construct of CNE (140)-actGFP used in (a); mt1, m2 and mt3 are point mutations (Msx (M), Tcf/Lef (T), and Jun/Fos (J)) generated from wt (140), and the detailed mutation design is shown in (b). The bar chart shows the percentage of the embryos that showed GFP expression in the neural plate among total developed embryos injected with the vector constructs. Actual numbers of GFP-positive cases and total numbers of scored embryos are indicated in parentheses. The chi-square test showed that the percentage of positive cases in the wt (140) and the Jun/Fos mutant constructs are significantly different (P < 0.0001), whereas the differences observed in other cases were not significant (P > 0.05)
Fig. 1. Kmer (k = 17) frequency analysis. a Kmer species frequency graph. The horizontal axis shows the depth of kmer species, and the vertical axis shows the percentage of each kmer species value (blue curve). b. Kmer individuals frequency graph. The horizontal axis shows depth of kmer individuals, the left vertical axis shows the percentage of each kmer individual’s value (blue curve), and the right vertical axis shows the accumulative frequency of the kmer individuals (red curve)
Fig. 2. One fosmid insert sequence of the gene Djth (DJF-016O13). This figure shows the alignment of genomic DNA and RNA sequencing reads to one fosmid insert sequence of the gene Djth (DJF-016O13) and the repeated elements annotation of this fosmid sequence by Repbase. Green arrowheads show the10th and 11th exon of the Djth gene on the fosmid insert sequence. Grey spots show matches between the sequencing reads and the reference fosmid sequence, and black spots show mismatches. Red bars represent repetitive sequences
Fig. 3. D. japonica genome assembly workflow
Fig. 4. Alignment between the Djth fosmid insert sequence and its corresponding genome scaffold. In the illustration of the Djth fosmid structure, green arrowheads show the 10th and 11th exons of the Djth gene in the fosmid (see Fig. 2). In the illustration of the Djth fosmid repeated sequences, red bars represent repetitive sequences. In the illustration of the alignment between Djth fosmid and scaffold, pink blocks show matched sequences between the fosmid and the corresponding scaffold, while white blocks show mismatches, which were mainly caused by gaps in the scaffold
Fig. 5. Category of gene ontology annotation
Fig. 6. Conserved non-coding elements between D. japonica and S. mediterranea
Fig. 7. Transgenic experiments suggested that Djndk CNE3 might be a regulatory element. a The arrowhead shows CNE3 inserted actGFP vector, CNE (140)-actGFP driven GFP to express in the anterior region at the end of gastrulation in transgenic Xenopus embryos (n = 132). b Putative transcription factor-binding motifs are boxed in different colors; those subjected to mutation analysis are indicated by asterisks. The detailed point mutation design of three transcription factor-binding motifs are shown by red colored words. c Mutation analysis of CNE3 (the 140 bp element). actGFP is an empty reporter construct that contains the β-actin basal promoter; wt (140) is the construct of CNE (140)-actGFP used in (a); mt1, m2 and mt3 are point mutations (Msx (M), Tcf/Lef (T), and Jun/Fos (J)) generated from wt (140), and the detailed mutation design is shown in (b). The bar chart shows the percentage of the embryos that showed GFP expression in the neural plate among total developed embryos injected with the vector constructs. Actual numbers of GFP-positive cases and total numbers of scored embryos are indicated in parentheses. The chi-square test showed that the percentage of positive cases in the wt (140) and the Jun/Fos mutant constructs are significantly different (P < 0.0001), whereas the differences observed in other cases were not significant (P > 0.05)
Agata,
Brain regeneration from pluripotent stem cells in planarian.
2008, Pubmed
Agata,
Brain regeneration from pluripotent stem cells in planarian.
2008,
Pubmed Agata,
Structure of the planarian central nervous system (CNS) revealed by neuronal cell markers.
1998,
Pubmed Agata,
Regeneration and gene regulation in planarians.
2003,
Pubmed Agata,
Molecular and cellular aspects of planarian regeneration.
1999,
Pubmed Altschul,
Basic local alignment search tool.
1990,
Pubmed An,
A colony multiplex quantitative PCR-Based 3S3DBC method and variations of it for screening DNA libraries.
2015,
Pubmed Asami,
Cultivation and characterization of planarian neuronal cells isolated by fluorescence activated cell sorting (FACS).
2002,
Pubmed Boetzer,
Scaffolding pre-assembled contigs using SSPACE.
2011,
Pubmed Boetzer,
SSPACE-LongRead: scaffolding bacterial draft genomes using long read sequence information.
2014,
Pubmed Boetzer,
Toward almost closed genomes with GapFiller.
2012,
Pubmed Buels,
JBrowse: a dynamic web platform for genome visualization and analysis.
2016,
Pubmed Cantarel,
MAKER: an easy-to-use annotation pipeline designed for emerging model organism genomes.
2008,
Pubmed Cebrià,
FGFR-related gene nou-darake restricts brain tissues to the head region of planarians.
2002,
Pubmed
,
Xenbase Chikhi,
Informed and automated k-mer size selection for genome assembly.
2014,
Pubmed Conesa,
Blast2GO: a universal tool for annotation, visualization and analysis in functional genomics research.
2005,
Pubmed Egger,
To be or not to be a flatworm: the acoel controversy.
2009,
Pubmed Gnerre,
High-quality draft assemblies of mammalian genomes from massively parallel sequence data.
2011,
Pubmed Grabherr,
Full-length transcriptome assembly from RNA-Seq data without a reference genome.
2011,
Pubmed Grohme,
The genome of Schmidtea mediterranea and the evolution of core cellular mechanisms.
2018,
Pubmed Haas,
De novo transcript sequence reconstruction from RNA-seq using the Trinity platform for reference generation and analysis.
2013,
Pubmed Hardison,
Conserved noncoding sequences are reliable guides to regulatory elements.
2000,
Pubmed Hayashi,
Isolation of planarian X-ray-sensitive stem cells by fluorescence-activated cell sorting.
2006,
Pubmed Hayashi,
Single-cell gene profiling of planarian stem cells using fluorescent activated cell sorting and its "index sorting" function for stem cell research.
2010,
Pubmed Hayashi,
Expression patterns of Xenopus FGF receptor-like 1/nou-darake in early Xenopus development resemble those of planarian nou-darake and Xenopus FGF8.
2004,
Pubmed
,
Xenbase Huang,
CAP3: A DNA sequence assembly program.
1999,
Pubmed Inoue,
Planarian shows decision-making behavior in response to multiple stimuli by integrative brain function.
2015,
Pubmed Inoue,
Clathrin-mediated endocytic signals are required for the regeneration of, as well as homeostasis in, the planarian CNS.
2007,
Pubmed Inoue,
Morphological and functional recovery of the planarian photosensing system during head regeneration.
2004,
Pubmed Jurka,
Repbase Update, a database of eukaryotic repetitive elements.
2005,
Pubmed Kent,
BLAT--the BLAST-like alignment tool.
2002,
Pubmed Kroll,
Transgenic Xenopus embryos from sperm nuclear transplantations reveal FGF signaling requirements during gastrulation.
1996,
Pubmed
,
Xenbase Kurtz,
Versatile and open software for comparing large genomes.
2004,
Pubmed Langmead,
Ultrafast and memory-efficient alignment of short DNA sequences to the human genome.
2009,
Pubmed Levy,
Enrichment of regulatory signals in conserved non-coding genomic sequence.
2001,
Pubmed Luo,
SOAPdenovo2: an empirically improved memory-efficient short-read de novo assembler.
2012,
Pubmed Marçais,
A fast, lock-free approach for efficient parallel counting of occurrences of k-mers.
2011,
Pubmed Margulies,
Genome sequencing in microfabricated high-density picolitre reactors.
2005,
Pubmed Nadalin,
GapFiller: a de novo assembly approach to fill the gap within paired reads.
2012,
Pubmed Nakazawa,
Search for the evolutionary origin of a brain: planarian brain characterized by microarray.
2003,
Pubmed Nishimura,
Comparative transcriptome analysis between planarian Dugesia japonica and other platyhelminth species.
2012,
Pubmed Nishimura,
Regeneration of dopaminergic neurons after 6-hydroxydopamine-induced lesion in planarian brain.
2011,
Pubmed Nishimura,
Unusually Large Number of Mutations in Asexually Reproducing Clonal Planarian Dugesia japonica.
2015,
Pubmed Nishimura,
gVolante for standardizing completeness assessment of genome and transcriptome assemblies.
2017,
Pubmed Ogino,
Convergence of a head-field selector Otx2 and Notch signaling: a mechanism for lens specification.
2008,
Pubmed
,
Xenbase Okamoto,
Neural projections in planarian brain revealed by fluorescent dye tracing.
2005,
Pubmed Portales-Casamar,
JASPAR 2010: the greatly expanded open-access database of transcription factor binding profiles.
2010,
Pubmed Quinlan,
BEDTools: a flexible suite of utilities for comparing genomic features.
2010,
Pubmed Robb,
SmedGD: the Schmidtea mediterranea genome database.
2008,
Pubmed Ruan,
Pseudo-Sanger sequencing: massively parallel production of long and near error-free reads using NGS technology.
2013,
Pubmed Sánchez Alvarado,
Double-stranded RNA specifically disrupts gene expression during planarian regeneration.
1999,
Pubmed Shibata,
Inheritance of a Nuclear PIWI from Pluripotent Stem Cells by Somatic Descendants Ensures Differentiation by Silencing Transposons in Planarian.
2016,
Pubmed Shimoyama,
Multiple Neuropeptide-Coding Genes Involved in Planarian Pharynx Extension.
2016,
Pubmed Skinner,
JBrowse: a next-generation genome browser.
2009,
Pubmed Stanke,
AUGUSTUS: ab initio prediction of alternative transcripts.
2006,
Pubmed Wingender,
The TRANSFAC project as an example of framework technology that supports the analysis of genomic regulation.
2008,
Pubmed Xue,
L_RNA_scaffolder: scaffolding genomes with transcripts.
2013,
Pubmed Zerbino,
Velvet: algorithms for de novo short read assembly using de Bruijn graphs.
2008,
Pubmed Zhang,
The oyster genome reveals stress adaptation and complexity of shell formation.
2012,
Pubmed
