Preparation of developing Xenopus muscle for sarcomeric protein localization by high-resolution imaging

Abstract

Mutations in several sarcomeric proteins have been linked to various human myopathies. Therefore, having an in vivo developmental model available that develops quickly and efficiently is key for investigators to elucidate the critical steps, components and signaling pathways involved in building a myofibril; this is the pivotal foundation for deciphering disease mechanisms as well as the development of myopathy-related therapeutics. Although striated muscle cell culture studies have been extremely informative in providing clues to both the distribution and functions of sarcomeric proteins, myocytes in vivo develop in an irreproducible 3D environment. Xenopus laevis (frog) embryos are cost effective, compliant to protein level manipulations and develop relatively quickly (⩽ a week) in a petri dish, thus providing a powerful system for de novo myofibrillogenesis studies. Although fluorophore-conjugated phalloidin labeling is the gold standard approach for investigating actin-thin filament architecture, it is well documented that phalloidin-labeling can be challenging and inconsistent within Xenopus embryos. Therefore we highlight several techniques that can be utilized to preserve both antibody and fluorophore-conjugated phalloidin labeling within Xenopus embryos for high-resolution fluorescence microscopy.

Keywords: Immunofluorescence microscopy; Myofibrillogenesis; Phalloidin; Sarcomere; Xenopus laevis.

Figure 1. Somitic tissue dissecting procedure

The fixed stage 40 embryo is placed into a petri dish (a). With forceps, both the head (H) and tail (T) are removed (b). The yolk-filled belly region is separated from the somitic tissue (S) (c). The somites are split into two pieces (d). One somitic side will have the neural tube (NT) and notochord (NC) still attached and both structures are removed with forceps (e, f). The epidermis is peeled away from the somitic regions (g). The somitic tissue (S) is placed into a well formed from nail polish for immunostaining (h).

Figure 2. Cryosection preparation

A liquid nitrogen bath setup with an isopentane filled ladle (a). 4–5 embryos are cleared of all residual sucrose solution and covered with OCT freezing medium (b). The embryos are transferred to, and aligned in a cryomold (c) and then immersed into the isopentane bath (d) until they turn opaque (e). Samples are quickly allowed to dry, wrapped in aluminum foil and stored (e, f).

Figure 3. Setup for whole-mount immunohistochemistry (IHC), cryosection IHC and BABB preparation

For whole-mount IHC, the entire procedure is executed in a nail polish slide well (a). For cryosection IHC, sections on coverslips are incubated on rubber stoppers in a moist covered chamber (b). For BABB preparation of the somitic tissue, the isopropanol washes are carried out in parafilm wells (c, d).

Figure 4. Two different phalloidin staining patterns are observed in somitic tissue

Fluorescently-labeled phalloidin can exhibit two distinct staining patterns in the Xenopus muscle cells. Green arrows point to the Z-disk (Z) and the blue arrows point to the M-line (M) (c, d, g, h). For whole-mounts, phalloidin exhibits a staining pattern that spans the length of the actin-thin filament (b, d). Phalloidin will also exhibit a staining pattern that spans the length of the actin-thin filament except for a region within the I-band in close proximity to the Z-disc (f, h). With cryosections, fluorophore-conjugated phalloidins consistently stain the entire actin-thin filaments (Fig. 5, j–l). Scale bar is 5 µm.

Figure 5. Comparison of stains from whole-mount, cryosection and BABB approaches

When the epidermis is left on the whole-mount tissue, it obstructs the observation of myofibrils/sarcomeric proteins (a–c). Whole-mounts free of the epidermis exhibit staining for sarcomeric proteins (d–f) but yolk granules remain visible and obstruct subcellular structures (e, arrow). After staining, clearing somitic tissue with BABB masks yolk platelets; phalloidin labeling is preserved (g–i). Cryosections are superior for staining of actin filaments and sarcomeric proteins (j–l). Scale bar is 5 µm.