Identification and in vivo characterization of NvFP-7R, a developmentally regulated red fluorescent protein of Nematostella vectensis

Abstract

Background: In recent years, the sea anemone Nematostella vectensis has emerged as a critical model organism for comparative genomics and developmental biology. Although Nematostella is a member of the anthozoan cnidarians (known for producing an abundance of diverse fluorescent proteins (FPs)), endogenous patterns of Nematostella fluorescence have not been described and putative FPs encoded by the genome have not been characterized.

Methodology/principal findings: We described the spatiotemporal expression of endogenous red fluorescence during Nematostella development. Spatially, there are two patterns of red fluorescence, both restricted to the oral endoderm in developing polyps. One pattern is found in long fluorescent domains associated with the eight mesenteries and the other is found in short fluorescent domains situated between tentacles. Temporally, the long domains appear simultaneously at the 12-tentacle stage. In contrast, the short domains arise progressively between the 12- and 16-tentacle stage. To determine the source of the red fluorescence, we used bioinformatic approaches to identify all possible putative Nematostella FPs and a Drosophila S2 cell culture assay to validate NvFP-7R, a novel red fluorescent protein. We report that both the mRNA expression pattern and spectral signature of purified NvFP-7R closely match that of the endogenous red fluorescence. Strikingly, the red fluorescent pattern of NvFP-7R exhibits asymmetric expression along the directive axis, indicating that the nvfp-7r locus senses the positional information of the body plan. At the tissue level, NvFP-7R exhibits an unexpected subcellular localization and a complex complementary expression pattern in apposed epithelia sheets comprising each endodermal mesentery.

Conclusions/significance: These experiments not only identify NvFP-7R as a novel red fluorescent protein that could be employed as a research tool; they also uncover an unexpected spatio-temporal complexity of gene expression in an adult cnidarian. Perhaps most importantly, our results define Nematostella as a new model organism for understanding the biological function of fluorescent proteins in vivo.

Figure 1. Description of the endogenous fluorescence of Nematostella.

(A) Lateral and (B) oral views of a Nematostella adult under fluorescent illumination using GFP (green) and Texas Red (red) filters. The oral pole is indicated by an asterisk in A. (C–F) Decapitated oral poles of polyps viewed under white light and fluorescent illumination. The number of tentacles is indicated in each panel. To facilitate observation, the tentacles were surgically removed. (D) An aboral (inverted) view of the sample in C, clearly showing the eight endodermal mesenteries (m). (C′–F′) Show the pattern of the red fluorescence for each decapitated polyp, characterized by both short (s1–s8, white arrowhead) and long (green arrowhead) fluorescent domains. (G) Diagrammatic cross-section through the oral pole, summarizing the pattern of the red fluorescence in polyps with 12-, 14- and 16-tentacles, showing ectoderm (blue), endoderm (grey), and red fluorescence (red). The number of tentacles present in each radial segment is indicated between mesenteries, and an asterisk indicates the position of the siphonoglyph. The dashed line represents a virtual boundary separating the half of the animal associated with the siphonogliph pole and the opposite half. The scale bars are 1 mm and 0.5 mm in A and C, respectively.

Figure 2. Identification of a novel red fluorescent protein (NvFP-7R) in Nematostella.

(A) Drosophila S2 cells expressing NvFP-7R (scale bar = 25µm). (B) Purified protein solutions of NvFP-7R and DsRed. (C) Excitation and emission spectra of NvFP-7R. (D) Absorbance spectrum of NvFP-7R. (E) Phylogenetic tree showing the position of NvFP-7R with respect to the major anthozoan FP clades (colored clades: A, B, C and D) described in a previous report , . In our phylogenetic analysis, the chromo-red protein eforCP/RFP from Echinopora forskaliana seems to belong to the clade B. This tree was constructed by the neighbor-joining method with JTT matrix. Bootstrap percentages over 60% are shown on interior branches. Three Arthropod FPs are used as outgroup. The GenBank accession numbers for the protein names used in this phylogenetic analysis are described in .

Figure 3. Comparisons between the expression domains and fluorescent spectra of NvFP-7R and the endogenous red fluorescence (ERF).

(A and B) mRNA expression of nvfp-7r detected by in situ hybridization. Black arrows indicate representative domains of NvFP-7R expression. (C and D) The pattern of the endogenous red fluorescence. (A and C) Oral views. (B and D) lateral views. (E) Excitation and emission spectra of NvFP-7Rc and the endogenous red fluorescence. The scale bar is 0.5 mm.

Figure 4. Expression pattern of endogenous red fluorescence in the oral endoderm.

(A) Schematic drawing of Nematostella showing the position of the cross sections shown in the panels B, C and D. The oral-aboral axis is indicated. (B, C and D) Endogenous red fluorescence observed in increasingly aboral cross-sections through the oral pole. Nuclei are labelled with DAPI (blue). (E and E′) Pattern of red fluorescence in the folded region separating tentacles observed at the level of the cross section in the panel B. (F to G′) Pattern of red fluorescence in the mesentery at the level of C and D cross sections. Panels B, C, and D are projections of confocal z-stacks. The scale bars are 75µm and 10µm in B and E, respectively. Abbreviations: en, endoderm; ec, ectoderm; m, mesentery; ph, pharynx.

Figure 5. The orientation of the complementary expression domains of NvFP-7R in the two apposed cell layers forming each mesentery.

(A) Diagrammatic cross-section through the oral pole showing the expression pattern of NvFP-7R; B and C indicate the relative position of the confocal images shown in the corresponding panels. (B–B″) Z-projections of a cross section through the oral pole, revealing complementary expression of endogenous fluorescence of NvFP-7R (red) in the apposed epithelial comprising each mesentery. (C–C″) Detailed view of the complementary domains in an individual mesentery. Nuclei are labelled with DAPI (blue). The scale bars are 100µm and 25µm in B and C, respectively.

Figure 6. Summary of the expression pattern of the endogenous red fluorescence.

(A) Diagrammatic cross-section through the oral pole, showing the expression pattern of NvFP-7R in an adult animal at the 16-tentacle stage; B and C indicate the relative position of the confocal images shown in the corresponding panels. (B and B′) Z-projections of confocal images of a single mesentery. Fine cellular structures, presumably cilia, project from the apical side of the mesentery (white arrowhead). (C and C′) Cross-section through a single cell layer of the mesentery, showing intense NvFP-7R localization in contractile elements at the base of myo-epithelial cells (green arrowhead). (D–D″) Lateral view of the contractile elements at the base of myo-epithelial cells showing co-localization of the endogenous red fluorescence (red) and F-Actin (ACT, green). Scale bars are 25µm and 10µm in B and (C and D), respectively.