Characterization of differential transcript abundance through time during Nematostella vectensis development

BMC Genomics (Impact Factor: 3.99). 04/2013; 14(1):266. DOI: 10.1186/1471-2164-14-266
Source: PubMed


Nematostella vectensis, a burrowing sea anemone, has become a popular species for the study of cnidarian development. In previous studies, the expression of a variety of genes has been characterized during N. vectensis development with in situ mRNA hybridization. This has provided detailed spatial resolution and a qualitative perspective on changes in expression. However, little is known about broad transcriptome-level patterns of gene expression through time. Here we examine the expression of N. vectensis genes through the course of development with quantitative RNA-seq. We provide an overview of changes in the transcriptome through development, and examine the maternal to zygotic transition, which has been difficult to investigate with other tools.

We measured transcript abundance in N. vectensis with RNA-seq at six time points in development: zygote (2 hours post fertilization (HPF)), early blastula (7 HPF), mid-blastula (12 HPF), gastrula (24 HPF), planula (5 days post fertilization (DPF)) and young polyp (10 DPF). The major wave of zygotic expression appears between 7–12 HPF, though some changes occur between 2–7 HPF. The most dynamic changes in transcript abundance occur between the late blastula and early gastrula stages. More transcripts are upregulated between the gastrula and planula than downregulated, and a comparatively lower number of transcripts significantly change between planula and polyp. Within the maternal to zygotic transition, we identified a subset of maternal factors that decrease early in development, and likely play a role in suppressing zygotic gene expression. Among the first genes to be expressed zygotically are genes whose proteins may be involved in the degradation of maternal RNA.

The approach presented here is highly complementary to prior studies on spatial patterns of gene expression, as it provides a quantitative perspective on a broad set of genes through time but lacks spatial resolution. In addition to addressing the problems identified above, our work provides an annotated matrix that other investigators can use to examine genes and developmental events that we do not examine in detail here.

Download full-text


Available from: Rebecca Helm, Jan 08, 2015
  • [Show abstract] [Hide abstract]
    ABSTRACT: Changes in the expression and function of genes drive evolutionary change. Comparing how genes are regulated in different species is therefore becoming an important part of evo-devo studies. A key tool for investigating the regulation of genes is represented by bacterial artificial chromosomes (BAC)-reporter constructs. BACs are large insert libraries, often >100 kb, which thus capture the genomic sequences surrounding a gene of interest, including all, or nearly all, of the elements underpinning regulation. Recombinant BACs, containing a reporter gene in place of the endogenous coding sequence of genes, can be utilized to drive the expression of reporter genes under the regulatory control of the gene of interest while still embedded within its genomic context. Systematic deletions within the BAC-reporter construct can be used to identify the minimal reporter in an unbiased way, avoiding the risk of overlooking regulatory elements that may be many kilobases away from the transcription start-site. Nematostella vectensis (Edwardsiidae, Anthozoa, Cnidaria) has become an important model in regenerative biology, ecology, and especially in studies of evo-devo and gene-regulatory networks due to its interesting phylogenetic position and amenability to molecular techniques. The increasing interest in this rising model system also led to a demand for methods that can be used to study the regulation of genes in Nematostella. Here, we present our progress in employing BAC-reporter constructs to visualize gene-expression in Nematostella. Using a new Nematostella-specific recombination cassette, we made nine different BAC-reporter constructs. Although five BAC recombinants gave variable effects, three constructs, namely Nv-bra:eGFP::L10 BAC, Nv-dpp:eGFP::L10 BAC, and Nv-grm:eGFP::L10 BAC delivered promising results. We show that these three constructs express the reporter gene eGFP in 10.4-17.2% of all analyzed larvae, out of which 26.2-41.9% express GFP in a mosaic fashion within the expected domain. In addition to the expression within the known domains, we also observed cases of misexpression of eGFP and examples that could represent actual expression outside the described domain. Furthermore, we deep-sequenced and assembled five different BACs containing Nv-chordin, Nv-foxa, Nv-dpp, Nv-wnta, and Nv-wnt1, to improve assembly around these genes. The use of BAC-reporter constructs will foster cis-regulatory analyses in Nematostella and thus help to improve our understanding of the regulatory network in this cnidarian system. Ultimately, this will advance the comparison of gene-regulation across species and lead to a much better understanding of evolutionary changes and novelties.
    Integrative and Comparative Biology 08/2013; 53(5). DOI:10.1093/icb/ict091 · 2.93 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The lined sea anemone Edwardsiella lineata is an informative model system for evolutionary-developmental studies of parasitism. In this species, it is possible to compare alternate developmental pathways leading from a larva to either a free-living polyp or a vermiform parasite that inhabits the mesoglea of a ctenophore host. Additionally, E. lineata is confamilial with the model cnidarian Nematostella vectensis, providing an opportunity for comparative genomic, molecular and organismal studies.Description: We generated a reference transcriptome for E. lineata via high-throughput sequencing of RNA isolated from five developmental stages (parasite; parasite-to-larva transition; larva; larva-to-adult transition; adult). The transcriptome comprises 90,440 contigs assembled from >15 billion nucleotides of DNA sequence. Using a molecular clock approach, we estimated the divergence between E. lineata and N. vectensis at 215-364 million years ago. Based on gene ontology and metabolic pathway analyses and gene family surveys (bHLH-PAS, deiodinases, Fox genes, LIM homeodomains, minicollagens, nuclear receptors, Sox genes, and Wnts), the transcriptome of E. lineata is comparable in depth and completeness to N. vectensis. Analyses of protein motifs and revealed extensive conservation between the proteins of these two edwardsiid anemones, although we show the NF-kappaB protein of E. lineata reflects the ancestral structure, while the NF-kappaB protein of N. vectensis has undergone a split that separates the DNA-binding domain from the inhibitory domain. All contigs have been deposited in a public database (EdwardsiellaBase), where they may be searched according to contig ID, gene ontology, protein family motif (Pfam), enzyme commission number, and BLAST. The alignment of the raw reads to the contigs can also be visualized via JBrowse. The transcriptomic data and database described here provide a platform for studying the evolutionary developmental genomics of a derived parasitic life cycle. In addition, these data from E. lineata will aid in the interpretation of evolutionary novelties in gene sequence or structure that have been reported for the model cnidarian N. vectensis (e.g., the split NF-kappaB locus). Finally, we include custom computational tools to facilitate the annotation of a transcriptome based on high-throughput sequencing data obtained from a "non-model system."
    BMC Genomics 01/2014; 15(1):71. DOI:10.1186/1471-2164-15-71 · 3.99 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Understanding how new cell types arise is critical for understanding the evolution of organismal complexity. Questions of this nature, however, can be difficult to answer due to the challenge associated with defining the identity of a truly novel cell. Cnidarians (anemones, jellies, and their allies) provide a unique opportunity to investigate the molecular regulation and development of cell-novelty because they possess a cell that is unique to the cnidarian lineage and that also has a very well-characterized phenotype: the cnidocyte (stinging cell). Because cnidocytes are thought to differentiate from the cell lineage that also gives rise to neurons, cnidocytes can be expected to express many of the same genes expressed in their neural "sister" cells. Conversely, only cnidocytes posses a cnidocyst (the explosive organelle that gives cnidocytes their sting); therefore, those genes or gene-regulatory relationships required for the development of the cnidocyst can be expected to be expressed uniquely (or in unique combination) in cnidocytes. This system provides an important opportunity to: (1) construct the gene-regulatory network (GRN) underlying the differentiation of cnidocytes, (2) assess the relative contributions of both conserved and derived genes in the cnidocyte GRN, and (3) test hypotheses about the role of novel regulatory relationships in the generation of novel cell types. In this review, we summarize common challenges to studying the evolution of novelty, introduce the utility of cnidocyte differentiation in the model cnidarian, Nematostella vectensis, as a means of overcoming these challenges, and describe an experimental approach that leverages comparative tissue-specific transcriptomics to generate hypotheses about the GRNs underlying the acquisition of the cnidocyte identity.
    Integrative and Comparative Biology 04/2014; 54(4). DOI:10.1093/icb/icu027 · 2.93 Impact Factor
Show more