Refinement of innervation accuracy following initial targeting of peripheral gustatory fibers

Abstract

During development, axons of the chorda tympani nerve navigate to fungiform papillae where they penetrate the lingual epithelium, forming a neural bud. It is not known whether or not all chorda tympani axons initially innervate fungiform papillae correctly or if mistakes are made. Using a novel approach, we quantified the accuracy with which gustatory fibers successfully innervate fungiform papillae. Immediately following initial targeting (E14.5), innervation was found to be incredibly accurate: specifically, 94% of the fungiform papillae on the tongue are innervated. A mean of five papillae per tongue were uninnervated at E14.5, and the lingual tongue surface was innervated in 17 places that lack fungiform papillae. To determine if these initial errors in papillae innervation were later refined, innervation accuracy was quantified at E16.5 and E18.5. By E16.5 only two papillae per tongue remained uninnervated. Innervation to inappropriate regions was also removed, but not until later, between E16.5 and E18.5 of development. Therefore, even though gustatory fibers initially innervate fungiform papillae accurately, some errors in targeting do occur that are then refined during later embryonic periods. It is likely that trophic interactions between gustatory neurons and developing taste epithelium allow appropriate connections to be maintained and inappropriate ones to be eliminated.

Figure 1

As the tongue grows from E14.5 to E18.5 of development, fungiform papillae undergo substantial changes in morphology. Increases in tongue size at E14.5 (A), E16.5 (B), and E18.5(C) are depicted using SEM. At higher magnification (1770×), developmental changes in surface papilla morphology can be observed at E14.5 (D), E16.5 (E), and E18.5 (F). No changes occur in the total number of fungiform papillae on the dorsal tongue surface between E14.5 and E18.5 of development (G). The scale bar in (C) = 600 μm and applies to (A–C). The scale bar in (F) = 10 μm and applies to (D–F).

Figure 2

At E14.5 chorda tympani fibers branch extensively and change trajectory numerous times between where they enter the tongue and fungiform papillae. A side view of an entire E14.5 tongue (A) showing the initial branches exiting the chorda tympani at the tongue base, these fiber bundles branch again as they near the surface, and these branches extend parallel to the tongue surface. Fiber bundles at the tongue surface appear to project to specific locations within the lingual epithelium. Confocal images of 50 μm vibrating microtome sections (B,C) provide another view of these branching characteristics. Large fiber bundles project from the tongue base to the tongue surface (B), and nearer the epithelial surface these fiber bundles branch, extending parallel to the surface (C). Fiber bundles near the epithelial surface project to individual fungiform papillae (C). Fiber bundles defasciculate at the epithelial-lamina propria border [(D,E), arrowheads], forming a very characteristic bud-shaped termination near the surface of the epithelium. These neural bud endings can be easily observed from the surface in tongue whole-mounts [(F), white arrows]. This dorsal surface view also clearly demonstrates the branching pattern of these fiber bundles parallel to the epithelium (F). Scale bar in (A) = 100 μm. Scale bar in (C) = 100 μm and applies to (B) and (C). Scale bar in (F) = 100 μm.

Figure 3

SEM images of the tongue can be aligned with DiI-labeled images of the same tongue at E16.5 (A–C) and E14.5 (D–F) to quantify innervation to individual fungiform papillae. Individual fungiform papillae are identified in SEM images (A,D). While most fungiform papillae are innervated at E14.5 and E16.5, one papilla on the imaged tongue is not innervated at each age [(B,E), arrows]. In addition, there were regions on the tongue surface that appeared to be innervated where no fungiform papillae were observed [(C,F), arrowheads]. Scale bar in (C) = 100 μm and applies to (A,–C). Scale bar in (F) = 100 μm and applies to (D–F).

Figure 4

A few chorda tympani fiber bundles project toward the epithelial surface without penetrating it at E14.5 [(A,B), arrows], and a few chorda tympani fiber bundles penetrate the epithelium in locations where no fungiform papillae are evident at E14.5 [(C), arrows] and E16.5 [(D,E), arrows]. Scale bar = 20 μm and applies to all.