Fixation of Embryonic Mouse Tissue for Cytoneme Analysis

Abstract

Developmental tissue patterning and postdevelopmental tissue homeostasis depend upon controlled delivery of cellular signals called morphogens. Morphogens act in a concentration- and time-dependent manner to specify distinct transcriptional programs that instruct and reinforce cell fate. One mechanism by which appropriate morphogen signaling thresholds are ensured is through delivery of the signaling proteins by specialized filopodia called cytonemes. Cytonemes are very thin (≤200 nm in diameter) and can grow to lengths of several hundred microns, which makes their preservation for fixed-image analysis challenging. This paper describes a refined method for delicate handling of mouse embryos for fixation, immunostaining, and thick sectioning to allow for visualization of cytonemes using standard confocal microscopy. This protocol has been successfully used to visualize cytonemes that connect distinct cellular signaling compartments during mouse neural tube development. The technique can also be adapted to detect cytonemes across tissue types to facilitate the interrogation of developmental signaling at unprecedented resolution.

Figure 1:. Example of E9.5 Shh-Cre; Rosa26 mTmG stained, embedded, and sectioned embryo.

(A) A single embryo in a 12-well plate, embedded in 4% LMP agarose. (B) Example of a correctly oriented embryo within the agarose block cut down to size for vibratome mounting. Excess agarose block is present along the bottom. (C) Bright field image of 100 μm thick embryo section embedded in LMP agarose. (D) Immunofluorescence imaging of a section after removal of agarose. Anti-GFP-stained mGFP (green) represents Shh-expressing cells in the tissue, with other cell lineages expressing membrane Tomato (red), DAPI in blue. Neural tube (bracket) and mGFP-positive notochord (arrow) are clearly visible. Scale bars = 1 cm (A), 5 mm (B), and 100 μm (C,D). Abbreviations: LMP = low melting point; mGFP = membrane green fluorescent protein; Shh = Sonic Hedgehog; DAPI = 4’,6-diamidino-2-phenylindole.

Figure 2:. Example of cryostat-sectioned neural tube floor plate and notochord with fragmented membrane extensions.

(A) 20 μm thick E9.5 Shh-Cre; Rosa26 mTmG^– section stained for GFP (green), F-actin (red), and DAPI (blue). mGFP puncta are visible between the notochord and floorplate (arrowhead) and (B,B’) migrating between adjacent cells of the neural tube. (C,C’) F-actin staining fails to detect any clear cytonemes on mesenchymal cells surrounding the neural tube (arrow). Scale bars = 20 μm. Abbreviations: mGFP = membrane green fluorescent protein; Shh = Sonic Hedgehog; DAPI = 4’,6-diamidino-2-phenylindole.

Figure 3:. Examples of optimal and suboptimal vibratome tissue sections and staining of the neural tube floor plate, notochord, and surrounding cells.

Examples of delicately handled sections (A,C), compared to (B) a folded tissue section and (D) a poorly handled section from a Shh-Cre; Rosa26 mTmG and a Shh^GFP/+ embryo^–. Optimally handled sections allow detection of cytonemes between adjacently localized floor plate neural epithelial cells (A, arrowheads). Neural tube adjacent notochord (A, mGFP-expressing) and mesenchymal cells (A, arrow) should be visible. (C,D) F-actin- and DAPI-stained sections should have consistent spacing of mesenchymal cells and F-actin-based cytonemes (arrows) surrounding the neural tube and notochord (C). Any minor folding or disruption of the sections can cause gaps and broken F-actin fragments (D, arrowheads). Scale bars = 10 μm. Abbreviations: mGFP = membrane green fluorescent protein; Shh = Sonic Hedgehog; DAPI = 4’,6-diamidino-2-phenylindole.