Graded and discontinuous EphA-ephrinB expression patterns in the developing auditory brainstem

Abstract

Eph-ephrin interactions guide topographic mapping and pattern formation in a variety of systems. In contrast to other sensory pathways, their precise role in the assembly of central auditory circuits remains poorly understood. The auditory midbrain, or inferior colliculus (IC) is an intriguing structure for exploring guidance of patterned projections as adjacent subdivisions exhibit distinct organizational features. The central nucleus of the IC (CNIC) and deep aspects of its neighboring lateral cortex (LCIC, Layer 3) are tonotopically-organized and receive layered inputs from primarily downstream auditory sources. While less is known about more superficial aspects of the LCIC, its inputs are multimodal, lack a clear tonotopic order, and appear discontinuous, terminating in modular, patch/matrix-like distributions. Here we utilize X-Gal staining approaches in lacZ mutant mice (ephrin-B2, -B3, and EphA4) to reveal EphA-ephrinB expression patterns in the nascent IC during the period of projection shaping that precedes hearing onset. We also report early postnatal protein expression in the cochlear nuclei, the superior olivary complex, the nuclei of the lateral lemniscus, and relevant midline structures. Continuous ephrin-B2 and EphA4 expression gradients exist along frequency axes of the CNIC and LCIC Layer 3. In contrast, more superficial LCIC localization is not graded, but confined to a series of discrete ephrin-B2 and EphA4-positive Layer 2 modules. While heavily expressed in the midline, much of the auditory brainstem is devoid of ephrin-B3, including the CNIC, LCIC Layer 2 modular fields, the dorsal nucleus of the lateral lemniscus (DNLL), as well as much of the superior olivary complex and cochlear nuclei. Ephrin-B3 LCIC expression appears complementary to that of ephrin-B2 and EphA4, with protein most concentrated in presumptive extramodular zones. Described tonotopic gradients and seemingly complementary modular/extramodular patterns suggest Eph-ephrin guidance in establishing juxtaposed continuous and discrete neural maps in the developing IC prior to experience.

Keywords: Cochlear nucleus; Eph receptor; Ephrin; Inferior colliculus; Lateral lemniscus; Superior olivary complex; Topography.

Figure 1

Early neonatal IC X-Gal labeling for ephrin-B2 (A) and EphA4 (B) at P8. Brightness profiles generated via rectangular (CNIC; C, D) and curved contour (LCIC Layer 3; E, F) sampling using ImageJ software. Areas of high protein expression and dark reaction product yield lower brightness values, whereas regions with low protein expression and less X-Gal staining correspond to higher brightness values. Linear regressions show similar slopes/gradients in ephrin-B2 (C) and EphA4 (D) CNIC expression. LCIC Layer 3 brightness plots reveal ephrin-B2 (E) and EphA4 (F) gradients comparable to those observed in the CNIC. In both instances, expression gradients followed known frequency axes (CNIC: ventromedial-to-dorsolateral, LCIC Layer 3: ventral-to-dorsal), with protein levels most concentrated in high-frequency regions. Scale bars = 200 μm.

Figure 2

Discontinuous ephrin-B2 (A) and EphA4 (B) LCIC Layer 2 expression during the first postnatal week (P8). Curved sampling through LCIC Layer 2 (solid contours in A, B) reveals periodic ephrin-B2 and EphA4 modules (dashed contours in A, B; corresponding arrows in C, D plot profiles). Scale bars = 200 μm.

Figure 3

CNIC (A) and LCIC Layer 2 (C) ephrin-B3 X-Gal labeling at P8 with corresponding brightness profiles (B, D; respectively). Rectangular sampling and linear regression analysis of CNIC (B) show an absence of ephrin-B3 protein prior to hearing onset. While positive in extramodular regions, curved contour sampling along LCIC Layer 2 reveal distinct ephrin-B3-negative modules (arrows in D). Ephrin-B3 positive fibers are apparent coursing through the IC commisure (arrowheads in A, C). Scale bars = 200 μm.

Figure 4

Higher magnification LCIC expression of ephrin-B2 (A) EphA4 (B) and ephrin-B3 (C) at P8. Ephrin-B2 and EphA4 expression includes somata and neuropil labeling that is most heavily concentrated within periodic, discontinuous presumptive modular fields (A, B, dashed contours). In contrast, ephrin-B3 labeling is low in presumptive modular zones (C, dashed contours) and more uniformly distributed in surrounding extramodular domains. Scale bars = 50 μm.

Figure 5

Normalized linear regression plots of CNIC expression for compiled ephrin-B2 (A, B), EphA4 (C, D), and ephrin-B3 (E, F) data. Plots were grouped into two developmental categories (P0/P4: A, C, E and P8/P12: B, D, F). Clear CNIC gradients are observed for both ephrin-B2 (A, B) and EphA4 (C, D) at each of the defined developmental pairings. In contrast, ephrin-B3 (E, F) is conspicuously absent from the nascent CNIC.

Figure 6

Ventral and dorsal cochlear nuclei expression patterns at P4 for ephrin-B2 (A, B), EphA4 (C, D), and ephrin-B3 (E, F). Ephrin-B2 is uniformly expressed throughout the VCN and DCN, albeit lacking in the overlying molecular layer (A, B). EphA4 expression is not uniform, but graded, with protein increasingly concentrated in more dorsal, high-frequency regions (C, D; white arrows). Complementary to ephrin-B2, VCN and DCN are largely devoid of ephrin-B3 protein (E, F), aside from positive label confined to the molecular layer and nonuniform expression in the auditory nerve root (VIII). Dashed contours represent nuclear boundaries. Scale bars = 200 μm.

Figure 7

X-Gal staining of the superior olivary complex (SOC) for ephrin-B2 (A), EphA4 (B), and ephrin-B3 (C) at P4. A. Ephrin-B2 is uniformly expressed throughout the LSO, SPON, MNTB, and DPO. B. EphA4 exhibits similar expression in the LSO, SPON, and MNTB, as well as prominent labeling throughout the MVPO and LVPO. C. Noteworthy ephrin-B3 expression is lacking in the early postnatal SOC. DPO = dorsal periolivary nucleus, LSO = lateral superior olivary nucleus, LVPO = lateroventral periolivary nucleus, MNTB = medial nucleus of the trapezoid body, MVPO = medioventral periolivary nucleus, SPON = superior paraolivary nucleus. Dashed contours represent nuclear boundaries. Scale bars = 200 μm.

Figure 8

Ephrin-B2 (A), EphA4 (B), and ephrin-B3 (C) expression at P8 in the dorsal nucleus of the lateral lemniscus (DNLL, dashed contours). Ephrin-B2 and EphA4 patterns appear complementary to ephrin-B3 labeling, which is absent from the DNLL, as well as fibers of the lateral lemniscus and commissure of Probst (arrowheads in C). Dashed contours denote DNLL boundaries. Scale bars = 200 μm.

Figure 9

Complementary midline expression of ephrin-B2 (A), EphA4 (B), and ephrin-B3 (C) at P4. The dorsal midline is ephrin-B2-positive (arrow in A) as is the entire midline for ephrin-B3 (arrow in C), while devoid of EphA4 (arrow in B). IC commissural fibers are consistently positive for ephrin-B3 (arrowhead in C). cIC = commissure of the inferior colliculus, SC = superior colliculus. Scale bars = 500 μm.

Figure 10

Summary of ephrin-B2, EphA4, and ephrin-B3 auditory brainstem expression prior to experience. Continuous expression gradients along the tonotopic CNIC and LCIC Layer 3 are juxtaposed with discontinuous and complementary LCIC Layer 2 modular/extramodular expression. In addition to these three proteins, other members of the Eph-ephrin signaling family are likely involved in guiding continuous and discrete neural maps in the developing IC (see www.brain-map.org for in situ hybridization data). Considerable work remains identifying corresponding presynaptic guidance molecules and the precise mechanisms by which they influence IC projection mapping.