In vivo imaging of Nematostella vectensis embryogenesis and late development using fluorescent probes

Abstract

Background: Cnidarians are the closest living relatives to bilaterians and have been instrumental to studying the evolution of bilaterian properties. The cnidarian model, Nematostella vectensis, is a unique system in which embryology and regeneration are both studied, making it an ideal candidate to develop in vivo imaging techniques. Live imaging is the most direct way for quantitative and qualitative assessment of biological phenomena. Actin and tubulin are cytoskeletal proteins universally important for regulating many embryological processes but so far studies in Nematostella primarily focused on the localization of these proteins in fixed embryos.

Results: We used fluorescent probes expressed in vivo to investigate the dynamics of Nematostella development. Lifeact-mTurquoise2, a fluorescent cyan F-actin probe, can be visualized within microvilli along the cellular surface throughout embryonic development and is stable for two months after injection. Co-expression of Lifeact-mTurquoise2 with End-Binding protein1 (EB1) fused to mVenus or tdTomato-NLS allows for the visualization of cell-cycle properties in real time. Utilizing fluorescent probes in vivo helped to identify a concentrated 'flash' of Lifeact-mTurquoise2 around the nucleus, immediately prior to cytokinesis in developing embryos. Moreover, Lifeact-mTurquoise2 expression in adult animals allowed the identification of various cell types as well as cellular boundaries.

Conclusion: The methods developed in this manuscript provide an alternative protocol to investigate Nematostella development through in vivo cellular analysis. This study is the first to utilize the highly photo-stable florescent protein mTurquoise2 as a marker for live imaging. Finally, we present a clear methodology for the visualization of minute temporal events during cnidarian development.

Figure 1

Phylogenetic position and life-cycle of Nematostella vectensis . A) Cnidarians are one of four early-evolved animal phyla that have recently been placed as the sister taxa to Bilaterians. (Phylogeny based on Hejnol [10]). B) The biphasic life cycle of anthozoan cnidarians like Nematostella vectensis begins with an early cleavage program that leads to a hollow blastula. At this stage, cells colored yellow mark the future site of gastrulation and where the mouth will eventually form (red star). During invagination the endo-mesoderm is formed, resulting in a diploblastic animal. In the ectoderm (blue) at the aboral pole of the planula larva a sensory structure called the apical organ (AO) or apical tuft forms. Once the animal undergoes metamorphosis, the animal possesses tentacles (T) used for feeding and can produce gametes for microinjection

Figure 2

Localization of Lifeact-mTurquoise2, tdTomato-NLS and EB1-mVenus in early cleavage embryos. A) Messenger RNA for three different fluorescent probes was injected in pairs into embryos of Nematostella vectensis. B-D) In the same embryo Lifeact-mTurquoise2 is localized to cell boundaries and overlaps with tdTomato-NLS around the nucleus. E-G) In dividing cells EB1-mVenus is clearly localized at mitotic spindle fibers and centrosomes. Interestingly Lifeact-mTurquoise2 (E) exhibits a brighter fluorescent ring around the nucleus in a subset of cells (white arrowhead) that start to form centrosomes adjacent to the nucleus (G). Lifeact-mTurquoise2 is also visible as striations corresponding to the location of spindle fibers labeled with EB1-mVenus (white arrows in E and F). H-I) Double expression of Lifeact-mTurquoise2 and mVenus in Nematostella embryos shows high concentration of Lifeact-mTurquoise2 around the nucleus (H) compared to mVenus used as a control (I) (white arrowheads). J-K) Both Lifeact-mTurquoise2 and mVenus appear as striations with the same morphology as spindle fibers (white arrows). L) Quantification of fluorescent i3ntensity of Lifeact-mTurquoise2 and tdTomato-NLS in nuclei of early cleavage stage embryos (the x-axis is represented as a white line in D). M) Quantification of fluorescent intensity of Lifeact-mTurquoise2 and untagged mVenus in nuclei of early cleavage stage embryos (the x-axis is represented as a white line in H and I). See main text for a more detailed description. (Scale bar =10 μm in each image)

Figure 3

Lifeact-mTurquoise2 localizes to the nuclear boundary and exhibits a ‘flash’ of Lifeact during nuclear disassembly. A) Time series of a living cell undergoing cell division. Just prior to nuclear disassembly, accumulation of Lifeact is apparent (A2.0-6.0) and during prometaphase disappears with a ‘flash’ (A6.5-7.5). See main text for a more detailed description of the full cleavage cycle. Scale bar 10 μm. B) When the cell rounds up during metaphase-anaphase Lifeact-mTurquoise2 fluorescence appears to increase at the cell boundary through time (different time points are indicated by colored lines). The average profiles perpendicular to the plasma membrane region going from the intracellular space (left) to the extracellular space (right) from the region depicted in A8.0 (yellow line) are normalized to the peak value of the profile at A8.0, show for this cell an increase of about 2.5 fold. C) Quantification of the Lifeact fluorescence at the nuclear boundary from 0.0-6.0 min. The profiles show the averaged normalized fluorescence perpendicular to the nuclear boundary going from nucleoplasm (left) to the cytoplasm (right)

Figure 4

Labeling of F-actin, DNA and tubulin in fixed embryos. A-B) Preserved cells labeled with the F-actin stain, phallicidin-FL (green) exhibit F-actin at the nuclear boundary of cells that are at an early phase of cell division. C) Alpha-tubulin staining (red) shows the formation of centrosomes adjacent to the nucleus. D) DNA staining with Hoechst (blue) show that the nuclear structures are still intact. E) Overlay of all three channels from B-D shows the relative position of all markers. F) Several cells at metaphase stained with the same labels show the fully formed mitotic spindles and the chromosomes that are lined up along the metaphase plate. F-actin associated with the nuclear boundary is no longer present, but is faintly visible in the cytoplasm and clearly at the plasma membrane

Figure 5

Localization of Lifeact-mTurquoise2 in microvilli and cell boundaries. A-B) Preserved embryos at early cleavage stages visualized using scanning electron microscopy, show microvilli on the cell surface. C) Similar stage preserved embryos stained using phallacidin to highlight filamentous-actin containing microvilli at the cells surface. D-E) During gastrulation, phallacidin can be used to visualize cellular boundaries along the outer surface of the gastrula. E) Zoomed in region of the ectoderm showing cellular boundaries. F-I) Cleavage stage embryos injected with the mRNA of Lifeact-mTurquoise2. F-G) Embryo approximately at the 32-cell stage, exhibits an increase in surface area contact between neighboring after cell cleavage. H-I) Embryo approximately at the 64-cell stage exhibits an increase in surface area contact between neighboring cells after cell cleavage. J-L) Gastrula stage embryos labeled through injection of Lifeact-mTurquoise2 RNA. J) Early gastrula where the endodermal plate is clearly visible by Lifeact-mTurquoise2 protein (red asterisk). K) Late gastrula where the endodermal plate has invaginated inward out of view (red asterisk – site of gastrulation). L) Magnification of cell boundaries clearly labeled with Lifeact-mTurquoise2 from late gastrulation stage embryo shown in K. M) Time series of an early embryo with a loose aggregate of cells with a flattened configuration that develops into a compact ball-shaped embryo, thereby increasing cell-cell contact (0.0-12.0 min, Additional file 7: Video S6)

Figure 6

Lifeact-mTurquoise2 protein can be used to identify many different structures in adult Nematostella vectensis . A) DIC image of a juvenile polyp approximately two weeks after fertilization. B) SEM image of the oral portion of the animal showing the ciliated cells at the surface. C) Laser confocal microscopy stack image of the aboral end of the polyp labeled with anti-alpha tubulin (secondarily labeled with Alexa 594) showing the tubulin that is present in ciliated cells. D) SEM image of the body wall of a polyp showing long cilia (white arrow) surrounded by ciliary cones. E) TEM image of a single central cilium with typical 9 + 2 arrangement of microtubules and nine stereocilia surrounding it. F) Body wall of a polyp originally injected during embryogenesis with Lifeact-mTurquoise2. Lifeact-mTurquoise2 highlights the stereocilia structures (white arrow) identified in D,E. G-H) Phallacidin labeled fixed polyp showing the intricate network of F-actin in the muscular tissue within the body (G) as well as cellular boundaries (H, white arrow). I) Single image from a time series of a moving polyp exhibiting an increased level of Lifeact-mTurquoise2 at the site of contraction (white arrows, also see Additional file 8: Video S7.) (J) Lifeact-mTurquoise2 also binds to the actin found in cell boundaries in polyps, similar to phallacidin (H). K) TEM image of cnidocyte (cn) with a curved nucleus at the basal pole (nu) showing filamentous actin-like fibers around the apex of cnidocyte capsule (inset). L) Prominent Lifeact-mTurquoise2 fluorescence is present around the apical and lateral regions of the cnidocytes, suggesting F-actin is present around the perimeter (white arrows)