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doi: 10.1371/journal.pone.0022725. Epub 2011 Jul 28.

Morphological and molecular analysis of the Nematostella vectensis cnidom

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Morphological and molecular analysis of the Nematostella vectensis cnidom

Claudia Zenkert et al. PLoS One.2011.

Abstract

The starlet sea anemone Nematostella vectensis is an emerging model organism for developmental and evolutionary biology. Due to the availability of genome data and its amenability to genetic manipulation Nematostella serves as a source for comparative molecular and phylogenetic studies. Despite this fact, the characterization of the nematocyst inventory and of nematocyst-specific genes is still fragmentary and sometimes misleading in this cnidarian species. Here, we present a thorough qualitative and quantitative analysis of nematocysts in Nematostella vectensis. In addition, we have cloned major nematocyst components, Nematostella minicollagens 1, 3 and 4, and show their expression patterns by in situ hybridization and immunocytochemistry using specific antibodies. Our data provides tools and insights for further studies on nematocyst morphogenesis in Nematostella and comparative evolution in cnidarians.

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Conflict of interest statement

Competing Interests:The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Capsule types inNematostella vectensis.
A–E. Basitrichous haplonemas. F–J. Microbasic mastigophores. K–P. Spirocysts. A, B. Light microscopic images of an undischarged (A) and a fully discharged (B) basitrichous haplonema. C–E. Scanning electron microscopic (SEM) images of haplonemas. C. Fully discharged haplonema. D. Detail of C, showing the basal spines (sp) and the opercular apparatus (op). E. Detail of the distal part of the tubule (t), revealing reduced spines (sp). F–J. Microbasic mastigophores. F, G. Undischarged (F) and fully discharged (G) capsule. H–J. SEM images of microbasic mastigophores. H. Fully discharged capsule. I. Detail of H showing the flexible spines (sp). J. Detail of the triangular tubule showing both the coiled and the distal uncoiled structure. K–P. Spirocysts. K–M. Light microscopic images of an undischarged (K), partly (L) and fully discharged spirocyst (M). N–P. SEM images of spirocysts. N. Undischarged spirocyst. O. Detail showing the closed opening of the capsule body. P. Distal end of discharged tubule. Scale bars are 10 µm (B, C, H, M), 5 µm (A, F, G, K, L, N), 1 µm (D, I, J, P), 300 nm (E), and 100 nm (O).
Figure 2
Figure 2. Quantitative distribution of nematocysts inNematostella polyps.
A. Distribution of nematocyst types in different developmental stages. B. Distribution of nematocyst types in different body parts of Nematostella vectensis.
Figure 3
Figure 3. Amino acid sequences ofNematostella minicollagens.
Protein sequences of minicollagens NvNCol-1, NvNCol-3, and NvNCol-4. Signal peptides in grey; propeptide in green; poly-proline stretches in pink; cysteine-rich domains (CRDs) are indicated in underlines, cysteine residues are in red. The C terminal CRDs printed in italics were chosen for raising the polyclonal antibodies.
Figure 4
Figure 4. Whole mountin situ hybridization for minicollagen genes at different developmental stages ofNematostella vectensis.
Gene expression patterns forNvNCol-1 (A–D),NvNCol-3 (E–H), andNvNCol-4 (I–L). Arrows in G denoteNvNCol-3 positive signals distributed along the body column. Dotted lines in H and L mark the borders of nematocytes showing positive signals. Scale bars are 100 µm (C, G and K), 50 µm (A, B, E, F, I and J), and 10 µm (D, H and L).
Figure 5
Figure 5. Detection ofNematostella minicollagens by immunocytochemistry.
Adult polyps were stained with polyclonal minicollagen antibodies and DAPI. A–D. NvNCol-1 staining for different capsule types. D shows a co-staining with NvNCol-4 antibody (green) to demonstrate a lack of NvCol-1 staining in developing spirocysts. E–H. staining for nematocysts using anti-NvNCol-3 antibody. I–L. NvNCol-4 staining of different nematocysts. In A–C and E–H minicollagens were stained with Alexa-568 (red) and in I–L with Alexa-488 (green). + positive signal. ++ strong signal. − no signal. Scale bars are 100 µm (A, E, and I), 10 µm (L), and 5 µm (B, C, D, F, G, H, J, and K).
Figure 6
Figure 6. Detection of minicollagens by Western blot analysis.
In each lane either isolated capsules or tentacle lysates of one adult head were applied. Preimmune serum (PPI) staining of tentacle lysates was used as negative control for each antibody. The asterisks mark the positions of the putative minicollagen monomer bands.
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