Characterizing the spatiotemporal expression of RNAs and proteins in the starlet sea anemone, Nematostella vectensis

Abstract

In an effort to reconstruct the early evolution of animal genes and proteins, there is an increasing focus on basal animal lineages such as sponges, cnidarians, ctenophores and placozoans. Among the basal animals, the starlet sea anemone Nematostella vectensis (phylum Cnidaria) has emerged as a leading laboratory model organism partly because it is well suited to experimental techniques for monitoring and manipulating gene expression. Here we describe protocols adapted for use in Nematostella to characterize the expression of RNAs by in situ hybridization using either chromogenic or fluorescence immunohistochemistry (∼1 week), as well as to characterize protein expression by whole-mount immunofluorescence (∼3 d). We also provide a protocol for labeling cnidocytes (∼3 h), the phylum-specific sensory-effector cell type that performs a variety of functions in cnidarians, including the delivery of their venomous sting.

Figure 1

Principal steps in chromogenic ISH and FISH. (a,b) Chromogenic ISH is shown in a, FISH in b. As described in the text, fluorescence is preferable for simultaneous detection of multiple RNA transcripts in the same cell. Both approaches use an antisense riboprobe in which some of the uridine residues are labeled with either digoxigenin (DIG) or fluorescein (FLU). Once the probe is bound to the target transcript, the digoxigenin or fluorescein molecules are then recognized by the appropriate antibodies. The antibodies are conjugated to either alkaline phosphatase (AP; in the case of chromogenic detection) or peroxidase (POD; in the case of fluorescence detection). In chromogenic detection, the presence of alkaline phosphatase is then visualized by BCIP and NBT. The alkaline phosphatase removes a phosphate group from BCIP, which is a colorless soluble compound. The BCIP then becomes oxidized in a reaction with NBT (a soluble yellow compound), which is simultaneously reduced. Both molecules precipitate out of the solution and assume a dark purple color. In fluorescence detection, numerous tyramide-conjugated fluorophores (e.g., Cy3 or fluorescein) aggregate in the vicinity of the bound RNA probe, as the tyramide is activated by the peroxidase (POD) bound to the antibody.

Figure 2

Example of ISH in Nematostella. (a–c) An antisense RNA probe for a single LWamide-like gene was detected using NBT/BCIP. The transcript is expressed in scattered individual cells in the outer ectodermal layer of the animal, as seen in deep (a) and superficial (b) focal planes in the embryo and in an intermediate plane in the juvenile polyp (c). (d,e) Antisense RNA probes for the same LWamide-like gene and a minicollagen gene (MiniCol3) were simultaneously detected in a larval anemone using fluorescence. The animals were imaged using confocal microscopy. Shown are the merged and individual channels. Each image represents a 10-μm z-projection. These images reveal clearly that the two transcripts are expressed in largely nonoverlapping cell populations. In all images, oral is to the left or indicated by an asterisk.

Figure 3

Example of indirect immunofluorescence. Whole-mount indirect immunofluorescence was performed with Nv-NF-κB-specific antiserum on a 4-week-old Nematostella polyp. Nv-NF-κB was detected with FITC-conjugated secondary antiserum. (a) Without AR. (b) With AR using citrate buffer. (c) With AR using urea. All images were taken using a FluoView FV10i confocal microscope with approximately the same settings for FITC (green) detection.

Figure 4

Example of cnidocyte staining. Nematostella juvenile polyps were fixed and stained under calcium-free conditions. (a) DAPI staining of the poly-γ-glutamate of cnidocyte capsules was detected in a green (521 nm) emission channel. (b) AO also stains cnidocyte capsules, and its fluorescence was detected in a red (615 nm) emission channel. In both stains, cnidocytes are primarily detected in the ectoderm of the body column.

Figure 5

Overview of the four protocol options described here. Steps 1A–C can be performed on Nematostella embryos, larvae or juvenile polyps. Cnidocytes cannot ordinarily be detected (Step 1D) in embryos or early larvae. A list of steps and timing for each element of each protocol option is provided.