Identification of gene regulatory elements in the sea anemone Nematostella vectensis

<p>The genetic complexity, genomic organization, and the level of sequence conservation in Nematostella vectensis, a cnidarian model organism, was found to be more similar to vertebrates than to other bilaterian model organisms. This lead to the assumption that the development of more complex body plans is mainly due to differences in gene regulation, rather than to different gene content between species.</p> <p>Cis-regulatory elements (CREs) (promoters, enhancers or silencers) are essential to regulate gene expression. They have been annotated in the genomes of bilaterian model organisms, but not in a single non-bilaterian metazoan genome.</p> <p>As gene regulatory mechanisms in Nematostella remain unknown, we set out to annotate promoters and enhancers in the Nematostella genome. To this end, we performed chromatin immunoprecipitation (ChIP) of the transcriptional coactivator p300, components of the basal transcription machinery (RNA Pol2) and several histone modifications in different developmental stages followed by Illumina sequencing.</p> <p>Quality of samples was assured and the locations of enrichments are conserved as expected and correlating with gene expression. We employed a supervised learning aproach using ChromHMM for prediction and annotation of putative CREs based on functional chromatin state segments genome-wide. Using this set, we validated some by testing CREs in vivo for their ability to drive expression of a reporter gene (shown in functional disection of Dpp region). The analysis of the different chromatin marks suggests that the genomic location, as well as the function of the different chromatin modifications in regulating gene expression, is conserved between bilaterian model organisms and Nematostella.</p> <p>Our genome-wide map of CREs in a basal metazoan reveals much similarity with bilaterians  and proves usefull in the study of the evolution of gene regulation in animals.</p> <p> </p> <p>Presented at the 2012 EvoNet Symposium in Vienna.</p

Nematostella vectensis transcriptome and gene models v2.0

<p>The gene models, transcripts and other derived data were generated from a comprehensive transcriptome survey of samples taken from five embryonic stages, adult female, and adult male samples. In total 1.6 billion 76-100 bp pair-end reads were obtained. The reads were mapped onto the N. vectensis genome v1.0, and new gene models were constructed using the RNA-seq evidence with the Augustus gene caller. These RNA-seq evidence based gene models are identified by a NVE prefix.</p

The Bilaterian Head Patterning Gene <em>six3/6</em> Controls Aboral Domain Development in a Cnidarian

<div><p>The origin of the bilaterian head is a fundamental question for the evolution of animal body plans. The head of bilaterians develops at the anterior end of their primary body axis and is the site where the brain is located. Cnidarians, the sister group to bilaterians, lack brain-like structures and it is not clear whether the oral, the aboral, or none of the ends of the cnidarian primary body axis corresponds to the anterior domain of bilaterians. In order to understand the evolutionary origin of head development, we analysed the function of conserved genetic regulators of bilaterian anterior development in the sea anemone <i>Nematostella vectensis</i>. We show that orthologs of the bilaterian anterior developmental genes <i>six3/6</i>, <i>foxQ2</i>, and <i>irx</i> have dynamic expression patterns in the aboral region of <i>Nematostella</i>. Functional analyses reveal that <i>NvSix3/6</i> acts upstream of <i>NvFoxQ2a</i> as a key regulator of the development of a broad aboral territory in <i>Nematostella</i>. <i>NvSix3/6</i> initiates an autoregulatory feedback loop involving positive and negative regulators of FGF signalling, which subsequently results in the downregulation of <i>NvSix3/6</i> and <i>NvFoxQ2a</i> in a small domain at the aboral pole, from which the apical organ develops. We show that signalling by <i>NvFGFa1</i> is specifically required for the development of the apical organ, whereas <i>NvSix3/6</i> has an earlier and broader function in the specification of the aboral territory. Our functional and gene expression data suggest that the head-forming region of bilaterians is derived from the aboral domain of the cnidarian-bilaterian ancestor.</p> </div

FGF receptor signalling suppresses <i>NvSix3/6</i> expression after gastrulation and is sufficient to induce apical organ formation.

<p>(A–D) Schematic representations of the morphological phenotype obtained after morpholino double injections. (D) Co-injection of <i>NvSix3/6</i> and <i>NvFGFa2</i> morpholinos reverses the loss of the apical tuft of <i>NvSix3/6</i> morphants (C). (E–P) Aboral views of in situ hybridizations at the midplanula stage (72 hpf); probes are indicated on the left side and morpholinos on top. Co-injection of <i>NvSix3/6</i> and <i>NvFGFa2</i> morpholinos leads to a moderate expansion in the expression of “spot genes” (E–L) and expansion of the aboral gap of the <i>NvSix3/6</i> expression (M–P). (Q–X) Effects of FGFR inhibitor on the double injected animals. Probes are indicated above the images, with morpholinos on the left side. Embryos were treated, from the late gastrula stage on, with DMSO (Q, R, U, V) or with the FGF receptor inhibitor SU5402 in DMSO (S, T, W, X). Inhibition of FGFR activity suppresses apical organ formation in double injected embryos. Scale bar, 100 µm.</p

A conserved anterior patterning system in <i>Nematostella</i> planula and sea urchin larva.

<p>(A and B) Model for gene regulatory network in place at gastrula and planula stages during aboral patterning of <i>Nematostella</i> larvae. At the gastrula stage (A), all the genes are co-expressed in the aboral area and <i>NvSix3/6</i> positively regulates the expression of FGF ligands and <i>NvFoxQ2a</i>. The positive regulation of <i>NvFGFa2</i> by <i>NvFGFa1</i> is already in place, and it is likely responsible for the early restriction of <i>FGFs</i>' expression domains. At the planula stage (B), the aboral territory is divided in two domains, one surrounding the apical organ (in purple and blue/red) and one in the apical organ (orange and yellow). The first corresponds to the expression domain of <i>NvSix3/6</i> and <i>NvFoxQ2a</i> (and <i>NvFoxD1</i>), the latter to the expression of FGF ligands (drawing). The regulatory interactions change at this stage and <i>NvFGFa1</i> represses <i>NvSix3/6</i>; additionally, <i>NvFGFa1</i> starts a positive autoregulation (direct or indirect), responsible for the maintenance of the <i>FGF</i> expressing area. (C) Segregation of expression domains in sea urchin larva. Similar to <i>Nematostell</i>a gastrula, the genes are co-expressed at the contra-blastoporal (anterior) pole of sea urchin (data from <i>Strongylocentrotus purpuratus</i> and <i>Paracentrotus lividus</i>) at early blastula and then segregate in distinct domains from the mesenchymal blastula stage on, when the tuft of cilia appears. In this case, however, the genes segregate in a slightly different way: <i>six3</i> forms a ring around the apical plate, while <i>foxQ2</i> is restricted to the apical plate itself. The apical domain expression of the FGF receptor is restricted to the apical plate.</p

<i>NvSix3/6</i> and <i>NvFGFRa</i> are required for the initiation of a FGF signalling feedback loop at gastrulation.

<p>(A–T) In situ hybridizations with probes indicated on top and injected morpholinos on the left. All animals are at the midgastrula stage (24 hpf); lateral views with aboral pole to the left are shown next to aboral views. In <i>NvSix3/6</i> morphants, the expression of <i>NvFGFa1</i> and <i>NvFGFa2</i> is reduced (C, D, G, H). In contrast to the situation at the planula stage, the expression of <i>NvFGFa2</i> but not <i>NvFGFa1</i> is reduced in <i>NvFGFa1</i> morphants (K, L). <i>NvFGFRa</i> is required for the expression of <i>NvFGFa1</i> and <i>NvFGFa2</i>, but not for <i>NvSix3/6</i> and <i>NvFoxQ2a</i> (Q–T). (U–W) Quantitative RT-PCR of (U) <i>NvSix3/6</i> MO-, (V) <i>NvFGFa1</i> MO-, and (W) <i>NvFGFa2</i> MO-injected embryos at the midgastrula stage (24 hpf). Fold changes of the relative expression levels of the indicated genes are shown; values between [−1, +1] mean no change, and +2 corresponds to 100% increase. Error bars represent the standard deviation of three biological replicates.</p

nveGenes.vienna130208.nemVec1.gtf

<p> </p> <p> </p> <p>Description: The gene models, transcripts and other derived data were generated from a comprehensive transcriptome survey of samples taken from five embryonic stages, adult femal, and adult male samples. In total 1.6 billion 76-100 bp pair-end reads were obtained. The reads were mapped onto the N. vectensis genome v1.0, and new gene models were constructed using the RNA-seq evidence with the Augustus gene caller. These RNA-seq evidence based gene models are identified by a NVE prefix.</p

nveGenes.vienna130208.nemVec1.bed

<p>Description: The gene models, transcripts and other derived data were generated from a comprehensive transcriptome survey of samples taken from five embryonic stages, adult femal, and adult male samples. In total 1.6 billion 76-100 bp pair-end reads were obtained. The reads were mapped onto the N. vectensis genome v1.0, and new gene models were constructed using the RNA-seq evidence with the Augustus gene caller. These RNA-seq evidence based gene models are identified by a NVE prefix.</p

<i>NvHoxF/Anthox1</i> and <i>NvSoxB(1)</i> have distinct roles in the regulation of aboral domain and apical organ genes.

<p>(A–C) Schematic drawings illustrating the morphology of <i>NvHoxF/Anthox1</i> (B) and <i>NvSoxB(1)</i> MO-injected planulae. Both genes are required for apical tuft formation, but only <i>NvSoxB(1)</i> morphants show a slightly elongated body column (C). (D–O) In situ hybridizations, lateral views (left) with the aboral pole to the left are shown next to aboral views (right) at the midplanula stage (72 hpf). In situ probes are indicated on the left, with morpholinos on top. <i>NvHoxF/Anthox1</i> and <i>NvSoxB(1)</i> are required for the expression <i>of NvFGFa1</i> (D–F) but not <i>NvFGFa2</i> (G–I). <i>NvSoxB(1)</i> morphants lack the gap in the expression of <i>NvSix3/6</i> and <i>NvFoxQ2a</i> (L, O), while <i>NvHoxF/Anthox1</i> morphants only lack the gap in <i>NvSix3/6</i> expression (K, N). Scale bar, 100 µm.</p

nveGenes.vienna130208.fasta

<p>Nematostella vectensis transcriptome and gene models v2.0</p> <p>Description: The gene models, transcripts and other derived data were generated from a comprehensive transcriptome survey of samples taken from five embryonic stages, adult femal, and adult male samples. In total 1.6 billion 76-100 bp pair-end reads were obtained. The reads were mapped onto the N. vectensis genome v1.0, and new gene models were constructed using the RNA-seq evidence with the Augustus gene caller. These RNA-seq evidence based gene models are identified by a NVE prefix.</p