Transcriptome characterization via 454 pyrosequencing of the annelid Pristina leidyi, an emerging model for studying the evolution of regeneration

Abstract

Background: The naid annelids contain a number of species that vary in their ability to regenerate lost body parts, making them excellent candidates for evolution of regeneration studies. However, scant sequence data exists to facilitate such studies. We constructed a cDNA library from the naid Pristina leidyi, a species that is highly regenerative and also reproduces asexually by fission, using material from a range of regeneration and fission stages for our library. We then sequenced the transcriptome of P. leidyi using 454 technology.

Results: 454 sequencing produced 1,550,174 reads with an average read length of 376 nucleotides. Assembly of 454 sequence reads resulted in 64,522 isogroups and 46,679 singletons for a total of 111,201 unigenes in this transcriptome. We estimate that over 95% of the transcripts in our library are present in our transcriptome. 17.7% of isogroups had significant BLAST hits to the UniProt database and these include putative homologs of a number of genes relevant to regeneration research. Although many sequences are incomplete, the mean sequence length of transcripts (isotigs) is 707 nucleotides. Thus, many sequences are large enough to be immediately useful for downstream applications such as gene expression analyses. Using in situ hybridization, we show that two Wnt/β-catenin pathway genes (homologs of frizzled and β-catenin) present in our transcriptome are expressed in the regeneration blastema of P. leidyi, demonstrating the usefulness of this resource for regeneration research.

Conclusions: 454 sequencing is a rapid and efficient approach for identifying large numbers of genes in an organism that lacks a sequenced genome. This transcriptome dataset will be a valuable resource for molecular analyses of regeneration in P. leidyi and will serve as a starting point for comparisons to non-regenerating naids. It also contributes significantly to the still limited genomic resources available for annelids and lophotrochozoans more generally.

Figure 1

Workflow of cDNA library construction. A mixed-stage regeneration/fission cDNA library was generated from ~4,500 P. leidyi worms. Anteriorly and posteriorly regenerating worms were collected from 0 to 3.5 days after amputation (dotted lines mark amputation planes; gray terminal masses represent regeneration blastemas) and actively fissioning worms were also collected (gray shading marks intercalated head and tail tissue that forms during fission). Following RNA extraction and cDNA synthesis, a portion of the pooled cDNA was normalized. The final library sent for 454 sequencing consisted of 2/3 normalized and 1/3 non-normalized cDNA.

Figure 2

Effectiveness of normalization of the cDNA library using duplex-specific nuclease. (A) Agarose gel smears show that non-normalized cDNA (treated with water) has distinct bands representing highly expressed genes, but these bands are absent in the normalized sample treated with duplex-specific nuclease (DSN). (B) RT-PCR analysis of transcript levels indicates that representation of the highly expressed genes Pl-β-actin and Pl-α-tubulin in the library is decreased after normalization with DSN. Representation of three lowly expressed genes, Pl-wnt-1, Pl-otx-2, and Pl-hox-Z, is increased, consistent with successful normalization. Standard error bars are shown.

Figure 3

Size distribution of largest isotig from each isogroup. A size distribution of the largest isotig from each isogroup shows that most isotigs are several hundred nucleotides in length, though some isotigs are as large as several thousand nucleotides.

Figure 4

Gene Ontology Biological Process designations of isotigs. Representative isotigs were subjected to Gene Ontology (GO) analysis using Blast2GO. Categories are level 2 Biological Process designations. Proportion on the y-axis was calculated from the total number of representative isotigs that were annotated with GO terms (11,140).

Figure 5

Validation of transcript assembly for two isogroups. Contigs and isotigs produced by the assembly are shown for two isogroups, (A) a putative piwi-like gene and (B) a putative frizzled gene. Blue boxes represent major contigs and V-shaped lines connect contigs that are adjacent to each other within the isotig. Short contigs of only a few nucleotides are omitted in this representation. Contigs that were independently confirmed by PCR and sequencing are represented as filled blue boxes (as opposed to open boxes) and connections between contigs that were independently confirmed by PCR and sequencing are indicated by solid V-shaped lines (as opposed to dotted lines). All major contigs and one isotig for each gene (isotigs 33900 (A) and 19226 (B)) were validated. Nucleotide sequence alignments are provided in Additional file 4.

Figure 6

Transcriptome coverage of four previously known gene sequences. All four developmental genes previously sequenced from P. leidyi are represented in the transcriptome, although coverage is incomplete. Black bars represent previously known sequence for each gene (GenBank numbers on left) and blue bars represent transcriptome sequences matching to or extending the reference sequence (isotig/contig numbers on left).

Figure 7

Expression of two Wnt/β-catenin pathway genes during regeneration and fission. Whole mount in situ hybridizations of Pl-fzA (A-E) and Pl-β-cat (F-J) show that both genes are expressed in the developing regeneration blastema as well as in new tissues forming by fission. (A-D) Following anterior amputation, Pl-fzA expression is not detectable (or only faintly so) before a blastema forms (A: 12 hours post amputation (hpa)), begins to be expressed in the early blastema (B: 1 day post amputation (dpa)) and persists through mid-stages of regeneration (C: 2 dpa). Pl-fzA is expressed in a similar fashion during posterior regeneration (D: 2 dpa). (F-I) Following anterior amputation, Pl-β-cat is similarly not detectable (or only faintly so) prior to blastema formation (F: 12 hpa), begins to be expressed in the early blastema (G: 1 dpa), and persists through mid-stages of regeneration (H: 3 dpa). Pl-β-cat is also expressed during posterior regeneration (I: 2 dpa). (E, J) During fission, both genes are expressed in developing fission zones (E, J: early fission - stage B), the transverse regions of tissue from which a new head and tail form (see Figure 1). All panels are lateral views with anterior to the left. Dark gray bars mark the extent of new tissue, i.e., the regeneration blastema or fission zone. Arrows point to early phase of expression of each gene on day 1.