Characterization of differential transcript abundance through time during Nematostella vectensis development.

BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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.

Developmental gene regulatory network connections predicted by machine learning from gene expression data alone.

Gene regulatory network (GRN) inference can now take advantage of powerful machine learning algorithms to complement traditional experimental methods in building gene networks. However, the dynamical nature of embryonic development-representing the time-dependent interactions between thousands of transcription factors, signaling molecules, and effector genes-is one of the most challenging arenas for GRN prediction. In this work, we show that successful GRN predictions for a developmental network from gene expression data alone can be obtained with the Priors Enriched Absent Knowledge (PEAK) network inference algorithm. PEAK is a noise-robust method that models gene expression dynamics via ordinary differential equations and selects the best network based on information-theoretic criteria coupled with the machine learning algorithm Elastic Net. We test our GRN prediction methodology using two gene expression datasets for the purple sea urchin, Stronglyocentrotus purpuratus, and cross-check our results against existing GRN models that have been constructed and validated by over 30 years of experimental results. Our results find a remarkably high degree of sensitivity in identifying known gene interactions in the network (maximum 81.58%). We also generate novel predictions for interactions that have not yet been described, which provide a resource for researchers to use to further complete the sea urchin GRN. Published ChIPseq data and spatial co-expression analysis further support a subset of the top novel predictions. We conclude that GRN predictions that match known gene interactions can be produced using gene expression data alone from developmental time series experiments.

Extending the family table: Insights from beyond vertebrates into the regulation of embryonic development by FGFs.

Since the discovery of fibroblast growth factors (FGFs) much focus has been placed on elucidating the roles for each vertebrate FGF ligand, receptor, and regulating molecules in the context of vertebrate development, human disorders and cancer. Studies in human, mouse, frog, chick, and zebrafish have made great contributions to our understanding of the role of FGFs in specific processes. However, in recent years, as more genomes are sequenced, information is becoming available from many non-vertebrate models and a more complete picture of the FGF superfamily as a whole is emerging. In some cases, less redundancy in these FGF signaling systems may allow for more mechanistic insights. Studies in sea anemones have highlighted how ancient FGF signaling is and helped provide insight into the evolution of the FGF gene family. Work in nematodes has shown that different splice forms can be used for functional specificity in invertebrate FGF signaling. Comparing FGFs between urochordates and vertebrates as well as between different insect species reveals important clues into the process of gene loss, duplication and subfunctionalization of FGFs throughout evolution. Finally, comparing all members of the FGF ligand superfamily reveals variability in many properties, which may point to a feature of FGFs as being highly adaptable with regards to protein structure and signaling mechanism. Further studies on FGF signaling outside of vertebrates is likely to continue to complement work in vertebrates by contributing additional insights to the FGF field and providing unexpected information that could be used for medical applications.

Analysis of Thisbe and Pyramus functional domains reveals evidence for cleavage of Drosophila FGFs.

BACKGROUND: As important regulators of developmental and adult processes in metazoans, Fibroblast Growth Factor (FGF) proteins are potent signaling molecules whose activities must be tightly regulated. FGFs are known to play diverse roles in many processes, including mesoderm induction, branching morphogenesis, organ formation, wound healing and malignant transformation; yet much more remains to be learned about the mechanisms of regulation used to control FGF activity. RESULTS: In this work, we conducted an analysis of the functional domains of two Drosophila proteins, Thisbe (Ths) and Pyramus (Pyr), which share homology with the FGF8 subfamily of ligands in vertebrates. Ths and Pyr proteins are secreted from Drosophila Schneider cells (S2) as smaller N-terminal fragments presumably as a result of intracellular proteolytic cleavage. Cleaved forms of Ths and Pyr can be detected in embryonic extracts as well. The FGF-domain is contained within the secreted ligand portion, and this domain alone is capable of functioning in the embryo when ectopically expressed. Through targeted ectopic expression experiments in which we assay the ability of full-length, truncated, and chimeric proteins to support cell differentiation, we find evidence that (1) the C-terminal domain of Pyr is retained inside the cell and does not seem to be required for receptor activation and (2) the C-terminal domain of Ths is secreted and, while also not required for receptor activation, this domain does plays a role in limiting the activity of Ths when present. CONCLUSIONS: We propose that differential protein processing may account for the previously observed inequalities in signaling capabilities between Ths and Pyr. While the regulatory mechanisms are likely complex, studies such as ours conducted in a tractable model system may be able to provide insights into how ligand processing regulates growth factor activity.

Genome-wide identification of enhancer elements.

We present a prospective genome-wide regulatory element database for the sea urchin embryo and the modified chromosome capture-related methodology used to create it. The method we developed is termed GRIP-seq for genome-wide regulatory element immunoprecipitation and combines features of chromosome conformation capture, chromatin immunoprecipitation, and paired-end next-generation sequencing with molecular steps that enrich for active cis-regulatory elements associated with basal transcriptional machinery. The first GRIP-seq database, available to the community, comes from S. purpuratus 24 hpf embryos and takes advantage of the extremely well-characterized cis-regulatory elements in this system for validation. In addition, using the GRIP-seq database, we identify and experimentally validate a novel, intronic cis-regulatory element at the onecut locus. We find GRIP-seq signal sensitively identifies active cis-regulatory elements with a high signal-to-noise ratio for both distal and intronic elements. This promising GRIP-seq protocol has the potential to address a rate-limiting step in resolving comprehensive, predictive network models in all systems.

Employing BAC-reporter constructs in the sea anemone Nematostella vectensis.

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.

A quantitative reference transcriptome for Nematostella vectensis early embryonic development: a pipeline for de novo assembly in emerging model systems.

BACKGROUND: The de novo assembly of transcriptomes from short shotgun sequences raises challenges due to random and non-random sequencing biases and inherent transcript complexity. We sought to define a pipeline for de novo transcriptome assembly to aid researchers working with emerging model systems where well annotated genome assemblies are not available as a reference. To detail this experimental and computational method, we used early embryos of the sea anemone, Nematostella vectensis, an emerging model system for studies of animal body plan evolution. We performed RNA-seq on embryos up to 24 h of development using Illumina HiSeq technology and evaluated independent de novo assembly methods. The resulting reads were assembled using either the Trinity assembler on all quality controlled reads or both the Velvet and Oases assemblers on reads passing a stringent digital normalization filter. A control set of mRNA standards from the National Institute of Standards and Technology (NIST) was included in our experimental pipeline to invest our transcriptome with quantitative information on absolute transcript levels and to provide additional quality control. RESULTS: We generated >200 million paired-end reads from directional cDNA libraries representing well over 20 Gb of sequence. The Trinity assembler pipeline, including preliminary quality control steps, resulted in more than 86% of reads aligning with the reference transcriptome thus generated. Nevertheless, digital normalization combined with assembly by Velvet and Oases required far less computing power and decreased processing time while still mapping 82% of reads. We have made the raw sequencing reads and assembled transcriptome publically available. CONCLUSIONS: Nematostella vectensis was chosen for its strategic position in the tree of life for studies into the origins of the animal body plan, however, the challenge of reference-free transcriptome assembly is relevant to all systems for which well annotated gene models and independently verified genome assembly may not be available. To navigate this new territory, we have constructed a pipeline for library preparation and computational analysis for de novo transcriptome assembly. The gene models defined by this reference transcriptome define the set of genes transcribed in early Nematostella development and will provide a valuable dataset for further gene regulatory network investigations.