Epithelial-derived brain-derived neurotrophic factor is required for gustatory neuron targeting during a critical developmental period

Abstract

Brain-derived neurotrophic factor (BDNF) is expressed in epithelial targets of gustatory neurons (i.e., fungiform papillae) before their innervation, and BDNF overexpression in nontaste regions of the tongue misdirects gustatory axons to these sites, suggesting that BDNF is necessary for gustatory axons to locate and innervate their correct targets during development. To test this hypothesis, we examined the targeting of taste neurons in BDNF-null mice (bdnf(-/-)). Analysis of bdnf(-/-) mice using a combination of DiI labeling and electron microscopy revealed that taste regions were not innervated by gustatory axons. Instead, branching was increased and many nontaste regions were innervated. The increased branching by gustatory axons in these animals was facilitated by neurotrophin 4 (NT4), because branching was virtually eliminated in bdnf(-/-)/nt4(-/-) mice. No abnormalities in gustatory innervation patterns and targeting were observed in nt4(-/-) mice. Conditional removal of BDNF selectively in epithelial cells disrupted targeting at the tongue tip, where gene recombination removed bdnf by embryonic day 13.5 (E13.5). However, innervation patterns were normal in the midregion and caudal portions of the tongue, where gene recombination did not occur until E14.5. These findings demonstrate that BDNF derived from gustatory epithelia is required for gustatory axons to correctly locate and innervate fungiform papillae. In addition, they show that BDNF-mediated targeting is restricted to a critical period of development, on or before E13.5.

Figure 1.

Innervation patterns do not differ between wild-type and bdnf ^−/− mice at E13.5. A–D, In wild-type (A, C) and bdnf ^−/− mice (B, D), DiI-labeled chorda tympani fibers branched near the tongue surface but did not reach it (most clearly seen in C). No neural buds were evident, and branching did not consistently extend the full medial-to-lateral width of the tongue surface. Individual tongues varied in the amount of branching. Orientation in A applies to B. R, Rostral; L, lateral. Orientation in C also applies to D. R, Rostral; D, dorsal. Scale bars: (in A) A, B, 100 μm; (in D) C, D, 100 μm.

Figure 2.

Chorda tympani fiber bundles fail to innervate fungiform papillae and undergo increased branching in bdnf ^−/− mice at E14.5. A–E, In wild-type mice, individual DiI-labeled chorda tympani fiber bundles showed a stereotypical pattern of innervation. Fiber bundles ended near the tongue surface in neural buds (A), which colocalized with fungiform papillae (SEM micrograph of papillae shown in B and DiI labeling/SEM micrograph overlay shown in C). The arrows in A–C show two examples of neural buds and corresponding fungiform papillae; a high-magnification view of these neural buds is shown in D. Innervation to the epithelial surface can be seen in cross section in E. F–J, In bdnf ^−/− mice, peripheral innervation patterns were disrupted. Neural buds were not clearly observed (F, high-magnification view of white box in I), although fungiform papillae were still present on the tongue; two examples are indicated with arrows (G). Papillae location did not correspond with the underlying innervation pattern (H). Branching was increased near the tongue surface (I; compared with D). Chorda tympani fiber bundles did not penetrate the epithelium in specific locations (J, arrows indicate the locations of papillae) as they did in wild-type mice (E) but innervate the epithelium randomly (J). Orientation in A applies to B–D and F–I. R, Rostral; L, lateral. Orientation in E also apples to J. R, Rostral; D, dorsal. Scale bars: (in F) A–C, F–H, 200 μm; (in I) D, I, 100 μm; (in J) E, J, 100 μm.

Figure 3.

Chorda tympani branching is increased in bdnf ^−/− mice. A–D, Branching of DiI-labeled fibers in the tongue midregion was more extensive in bdnf ^−/− mice (C, D) than in wild-type mice (A, B). Branching was quantified by tracing each fiber bundle (B, D). Branches that were connected within the sampled area are displayed in the same color. Scale bar: (in A) A–D, 100 μm. R, Rostral; L, lateral.

Figure 4.

Innervation in bdnf ^−/− tongues is lost at E16.5, although some fungiform papillae remain innervated. A, B, DiI-labeled chorda tympani fiber bundles branched elaborately at the surface of whole bdnf ^−/− tongues at E14.5 (A) and E16.5 (B). C–F, I, By E18.5, most surface innervation was withdrawn (C). A higher magnification view of the E18.5 tongue tip (F, which corresponds to boxed area in C) shows that fiber bundles ended in punctate regions. Because the depth of the tongue exceeds the focal depth of the microscope, innervation near the surface is in focus, whereas fibers deep within the tongue are not. Some fibers penetrated fungiform papillae (D, arrow), although aberrant innervation was present (E). I shows an SEM micrograph of the tongue in C. Although DiI-SEM overlays in these tongues were not possible, some innervated regions (F, arrows) corresponded to locations of fungiform papillae (I, arrows). G, H, Specific regions of the palate corresponding to the Geschmackssteifen (G, arrows). Specific regions of the posterior palatine field (G, arrowheads) were not innervated in bdnf ^−/− mice (H). This lack of specific innervation was apparent, although overall branching increased in the bdnf ^−/− palate (H) compared with the wild-type palate (G). Orientation in C applies to A–C and F–I. Orientation in E also applies to D. R, Rostral; D, dorsal; L, lateral. Scale bars: A–C, 500 μm; D, E, 100 μm; (in I) I, F, 100 μm; (in H) G, H, 500 μm.

Figure 5.

Most innervation of the tongue surface is lost in mice lacking both BDNF and NT4. A–C, DiI-labeled whole tongues from bdnf ^−/− (A), bdnf ^−/−/nt4 ^−/− (B), and nt4 ^−/− mice (C) at E15.5. D–I, DiI labeling of sagittal tongue sections. In bdnf ^−/− mice, substantial chorda tympani branching was present near the tongue surface, but fibers did not project to the specific regions of the tongue surface to form a neural buds (A, D, E). In bdnf ^−/−/nt4 ^−/− mice, most chorda tympani branches at the tongue surface were eliminated (B, F, G). The absence of branching at the tongue surface allows for visualization of the chorda tympani at the tongue base, which is completely obscured in bdnf ^−/− mice. A few fiber bundles (B, arrowheads) can be seen exiting the chorda tympani at the tongue base in bdnf ^−/−/nt4 ^−/− mice (B, arrows). Innervation was normal in nt4 ^−/− mice (C, H, I). Scale bars: (in C) A–C, 500 μm; (in H) D, F, H, 100 μm; (in I) E, G, I, 100 μm.

Figure 6.

Removal of NT4 does not disrupt targeting. A, B, Chorda tympani innervation patterns were generally similar in the tongues of wild-type (A) and nt4 ^−/− mice (B). C, E, Unlike in bdnf ^−/− tongues, clear neural buds can be seen at higher magnification in wild-type (arrows in C, which corresponds to white box in A) and nt4 ^−/− tongues (arrows in F, which corresponds to white box in B). D–H, These neural buds typically corresponded to fungiform papillae in SEM micrographs of the same wild-type (D, E, arrows) and nt4 ^−/− tongues (G, H, arrows). A few fungiform papilla were not associated with a neural bud (G, H, arrowhead). Scale bars: (in B) A, B, 500 μm; C–F, 200 μm.

Figure 7.

K14-Cre-induced Bdnf recombination must occur on or before E13.5 for targeting to be disrupted. A, D, Tongues from mice that express lacZ instead of BDNF after gene recombination. At E13.5, gene recombination had occurred at the tongue tip but was not complete in caudal portions of the tongue (A). At E14.5, X-Gal staining was robust in all fungiform papillae, indicating that gene recombination had occurred (D). B, E, G, H, DiI labeling of chorda tympani fibers in heterozygous BDNF knock-outs (bdnf^lox/neo) was normal. Neural buds were present on the dorsal surface (B, H, arrowheads). Fibers projected to individual fungiform papillae at the tongue tip (B, E, G, arrows). In conditional BDNF knock-outs (K14-bdnf ^lox/neo), innervation of the dorsal tongue surface by the chorda tympani was normal, with innervation to specific regions being apparent (C, J, arrowheads). However, at the tongue tip, branching increased and innervation was no longer specific (C, F, I, J). Scale bars: (in A) A, D, 200 μm; (in C) B, C, E, F, 250 μm; G (for G, I), H (for H, J), 100 μm.

Figure 8.

K14-Cre-induced recombination of both bdnf alleles disrupts innervation patterns at the tongue tip. A, B, DiI-labeled branching patterns in the dorsal tongue tip at E16.5. C, D, Branching patterns in the ventral tongue tip. E16.5 bdnf^lox/lox mice exhibited branching patterns in the dorsal (A) and ventral (C) tongue tip that were similar to those in wild-type animals (data not shown). Most chorda tympani fiber bundles ended in a clear neural bud near the tongue surface. In K14-Cre;bdnf^lox/lox mice, branching increased and the pattern of branching at the dorsal (B) and ventral (D) tongue tip was disrupted. Branching looked more like wild-type branching in caudal regions of the dorsal tongue tip, where some neural buds were evident (B, arrow). Scale bar: (in C) A–D, 250 μm.

Figure 9.

A diagram illustrating the role of neurotrophins in taste bud innervation. During development, the chorda tympani extends in a caudal-to-rostral manner without the support of neurotrophins (A). Either BDNF (blue dots) or NT4 (green dots) expressed by the tongue mesenchyme can support chorda tympani branching from the chorda tympani at the base of the tongue. Once fiber branches near the epithelial surface, they are attracted by BDNF-expressing fungiform placodes (blue half-circles). BDNF derived from fungiform papillae attracts chorda tympani fibers to the papilla epithelium and encourages them to penetrate the epithelial surface. Papilla innervation proceeds in a caudal-to-rostral manner, with more caudally located papillae being innervated earlier than more rostrally located papillae (B). The timing of epithelial maturation is independent of innervation. Specifically, keratin-14 expression (A, brown color) begins at the tongue tip and progresses caudally.