Profiling of maternal and developmental-stage specific mRNA transcripts in Atlantic halibut Hippoglossus hippoglossus

Abstract

cDNA libraries were constructed from the following developmental stages (tissues) of the Atlantic halibut (Hippoglossus hippoglossus): 2-cell stage (embryos), 1 day-old yolk sac larvae (trunk) and juvenile (fast skeletal muscle). A total of 4249 high quality expressed sequence tags from the three libraries were clustered into a partial transcriptome of 2124 putative genes. A large proportion of the gene clusters (48.3%) had no significant matches against known proteins. The most abundant ESTs of nuclear transcripts in the 2-cell library included sequences with high identity to zebrafish H1M, a linker histone-like protein involved in primordial germ cell specification, zinc finger protein, rRNA external transcribed spacer, thymosin β-4, cyclin B1 and several predicted peptides from the Tetraodon nigroviridis genome assembly with unknown functions. 170 and 123 ESTs represented ribosomal proteins in the larval and juvenile libraries respectively, compared with only two sequences in the 2-cell library, which may reflect an abundance of maternally inherited pre-formed ribosomes in the yolk. Even though some clusters were common to all three libraries, most putative genes showed a developmental-stage specific distribution with 72% (2-cell embryo), 59% (larval) and 57% (juvenile) sequences having no significant matches against the 8400 adult halibut sequences in the EMBL nucleotide database. Comparison between the predicted halibut peptide data set and the human, zebrafish, and pufferfishes (T. nigroviridis and Takifugu rubripes) proteomes revealed that, as expected, the halibut sequences were more similar to the other two fish species than to human proteins. However, no clear bias towards the pufferfishes was observed, suggesting significant sequence variation between orthologues within the clade Acanthomorpha. The sequence information generated in the present study will represent a significant new resource for future studies on normal and abnormal development in Atlantic halibut.

Introduction

The genomes of four model teleost fishes have been sequenced to the draft level: the zebrafish (Danio rerio), the medaka (Oryzias latipes) and the pufferfishes Takifugu rubripes and Tetraodon nigroviridis (http://www.ensemble.org/index.html). In contrast, large scale genetic resources for farmed fish species such as common carp (Cyprinus carpio), Atlantic salmon (Salmo salar), rainbow trout (Oncorhynchus mykiss) and tilapia (Oreochromis niloticus) are restricted to expressed sequence tags (ESTs), the product of high-throughput, single-pass cDNA sequence analyses (Cossins and Crawford, 2005).

The Atlantic halibut Hippoglossus hippoglossus (order Pleuronectiformes) is a valuable flatfish with an established market that is starting to be farmed commercially in Canada, Norway and Scotland (Bergh et al., 2001). A major bottleneck in halibut farming is the production of juveniles for on-growing. Significant problems include high embryonic and larval mortality and the prevalence of body deformities (Kjørsvik et al., 1990, Bromage et al., 1992). Paternity analysis using DNA microsatellite markers indicates that surviving juveniles typically come from a small percentage of the total broodstock population, indicating poor egg quality following artificial fertilization (Jackson et al., 2003). Abnormal patterns of blastomere cleavage are common and have been linked to low hatching success (Kjørsvik et al., 1990, Shields et al., 1997). Maternal mRNA transcripts and/or proteins have critical roles in directing early developmental processes in teleosts (Kane and Kimmel, 1993). In zebrafish, zygotic transcription only begins at the mid-blastula transition (512-cell stage) (Kane and Kimmel, 1993). In contrast, zygotic gene activation starts at the 1-cell stage in mouse embryos and is clearly evident by the 2-cell stage (Aoki et al., 1997, Zeng et al., 2004). During the maternal-to-zygotic transition oocyte-specific transcripts are degraded and maternal transcripts common to oocyte and early embryo are replaced with zygotic transcripts (Paynton et al., 1988, Davis et al., 1996, Zeng et al., 2004). Maternal mRNA transcripts and/or proteins are thought to control cell cleavage patterns, the establishment of body axes and specification of early embryonic cells prior to the mid-blastula phase (Nishikata et al., 2001, Yamada et al., 2005, Zeng et al., 2004, Zeng and Schultz, 2005). Post-ovulatory ageing in rainbow trout oocytes altered the abundance of mRNA transcripts, including insulin-like growth factor-I receptor, and was associated with developmental abnormalities in the larval stages (Aegerter et al., 2004).

Studies on developmental mechanisms in Atlantic halibut are hindered by a lack of molecular markers. All published EST datasets from Atlantic halibut are from the adult stage of the life cycle (e.g. Park et al., 2005) with a total of 8400 sequences deposited in the EMBL nucleotide data base. There have been no transcriptomal analyses concerned with skeletal muscle and/or early developmental stages and nothing is known about the composition of maternal mRNAs. The first aim of the present study was therefore to characterize cDNA libraries from 2-cell embryos, the trunk of yolk-sac larvae and the fast myotomal muscle of juveniles as a resource for future studies on normal and abnormal development.

The two pufferfish genomes sequenced to draft level belong to the same clade as the Atlantic halibut, the Acanthomorpha (ray finned fishes with true spines in their anal and dorsal fins) (Nelson, 1994). The Acanthomorpha represent nearly 60% of extant fish diversity with more than 15,300 species and 314 families (Nelson, 1988). Many of the nodes on the Acanthomorpha tree remain unresolved or poorly defined (Dettai and Lecointre, 2005). The second aim of the present study was to establish the extent to which genomic resources from model species, particularly pufferfishes, could be useful for studies with Atlantic halibut. We therefore investigated patterns of phylogenetic affinity between halibut EST sequences and the proteome databases available from pufferfish, zebrafish and human using the Java/Perl-based application SimiTri (Parkinson and Blaxter, 2003), which allowed simultaneous display and analysis of relative similarity relationships.