Time-dependent gene expression analysis of the developing superior olivary complex

Abstract

The superior olivary complex (SOC) is an essential auditory brainstem relay involved in sound localization. To identify the genetic program underlying its maturation, we profiled the rat SOC transcriptome at postnatal days 0, 4, 16, and 25 (P0, P4, P16, and P25, respectively), using genome-wide microarrays (41,012 oligonucleotides (oligos)). Differences in gene expression between two consecutive stages were highest between P4 and P16 (3.6%) and dropped to 0.06% between P16 and P25. To identify SOC-related genetic programs, we also profiled the entire brain at P4 and P25. The number of differentially expressed oligonucleotides between SOC and brain almost doubled from P4 to P25 (4.4% versus 7.6%). These data demonstrate considerable molecular specification around hearing onset, which is rapidly finalized. Prior to hearing onset, several transcription factors associated with the peripheral auditory system were up-regulated, probably coordinating the development of the auditory system. Additionally, crystallin-γ subunits and serotonin-related genes were highly expressed. The molecular repertoire of mature neurons was sculpted by SOC-related up- and down-regulation of voltage-gated channels and G-proteins. Comparison with the brain revealed a significant enrichment of hearing impairment-related oligos in the SOC (26 in the SOC, only 11 in the brain). Furthermore, 29 of 453 SOC-related oligos mapped within 19 genetic intervals associated with hearing impairment. Together, we identified sequential genetic programs in the SOC, thereby pinpointing candidates that may guide its development and ensure proper function. The enrichment of hearing impairment-related genes in the SOC may have implications for restoring hearing because central auditory structures might be more severely affected than previously appreciated.

Keywords: Auditory Processing Disorder; Circuit Development; Deafness; Genetic Diseases; Neurodevelopment; Neurodifferentiation; Neuroprotection; Retrocochlear Function; Transcription Factors.

FIGURE 1.

Hierarchical cluster analysis of the genome-wide expression patterns in the SOC and the entire brain. A, ontogeny of the rat SOC. Establishment of connectivity is completed by birth (P0), but pups are unable to hear until about P12. Functional and structural maturation of auditory circuits (e.g. synaptic refinement) takes place mainly during the first and second postnatal weeks. B, each row represents a distinct stage/tissue; each column represents a single oligo. Oligos are clustered according to the similarity of their normalized expression profiles. Color maps indicate a gene's expression level relative to the overall mean intensity of all investigated stages/tissues. Black, equal expression; green, higher expression; red, lower expression. The strongest similarity was observed between SOC-P16 and SOC-P25, and between SOC-P0 and SOC-P4. Most differences in expression levels occurred between pre- and posthearing stages. Br-P4 clustered together with prehearing SOC stages, whereas the mature age-matched stages showed a low degree of similarity, consistent with increasing specification during development.

FIGURE 2.

Validation of microarray data by qRT-PCR and RNA in situ hybridization. A, relative expression level changes of selected genes were examined by qRT-PCR to verify the microarray results. Consecutive developmental stages of the SOC were investigated. Black dashed lines, microarray data; gray dashed lines, qRT-PCR data of three biological replicates. Data sets were normalized to P0 = 1. *, p < 0.05; **, p < 0.01; ***, p < 0.001. Collectively, data sets from microarray experiments correlated strongly with those from qRT-PCR experiments. B, RNA in situ hybridization of coronal sections through the rat brainstem at P25–P30. Sections were hybridized with digoxigenin-labeled cRNA probe. Calb1 served as a positive control, and a sense probe served as negative control. All probes except for Calb1 hybridized throughout the SOC. The MNTB is always at the left-hand side, whereas the lateral superior olive is on the right-hand side. For orientation, a schema of the adult rat SOC is provided in the bottom right-hand corner. Shown are representative results from at least three independent hybridization experiments. LSO, lateral superior olive; LNTB, lateral nucleus of the trapezoid body; MSO, medial superior olive; SPN, superior paraolivary nucleus; VNTB, ventral nucleus of the trapezoid body. Scale bars (B), 200 μm.

FIGURE 3.

RNA in situ hybridization of SOC-related genes. RNA in situ hybridization of coronal sections through the mouse brainstem of P4 or P25 animals. Sections were hybridized with digoxigenin-labeled cRNA probes. All probes hybridized throughout the SOC. As a negative control, hybridization of a Cre-recombinase antisense probe is shown. Representative results from at least three independent hybridization experiments are shown. LSO, lateral superior olive. Scale bars, 200 μm.

FIGURE 4.

Expression profiles of genes that are highly expressed prior to hearing onset and display elevated expression in SOC-P4 compared with Br-P4. A–J, transcription factors; K–M, crystallin-γ subunits; N–P, serotonin signaling. Expression profiles are shown as dashed lines, and mean expression levels are normalized to SOC-P4 = 1. Dark gray, SOC development; light gray, brain development; black insets, comparison of age-matched SOC and brain at P4 and P25. *, p < 0.05; **, p < 0.01; ***, p < 0.001. Error bars, S.E.

FIGURE 5.

Crystallin-γ in the SOC. A, low magnification photomicrograph of the SOC-P5, demonstrating crystalline-γ immunoreactivity in the lateral superior olive (LSO), the medial superior olive (MSO), and the MNTB. Fibers in the ventral acoustic stria (arrowheads) and the trapezoid body medial to the MNTB (arrows) were also immunoreactive. B, high magnification photomicrograph of the trapezoid body, demonstrating the fascicular labeling pattern in fibers. C, lateral superior olive at higher magnification, with perisomatic labeling around spindle-shaped somata (most probably principal neurons). D, crystallin-γ in the SOC-P25. Compared with P5, immunoreactivity was lower throughout the SOC. E, high magnification photomicrograph of the trapezoid body medial to the MNTB, demonstrating fewer immunopositive fibers than at P5. F, MNTB at high magnification, demonstrating weakly immunopositive somata. G, lateral superior olive at high magnification, with a labeling pattern similar to that of P5. Dorsal is up and medial to the left in all panels. Scale bars, 200 μm (A), 250 μm (D), and 30 μm (B, C, and E–G).

FIGURE 6.

Expression profiles of selected gene categories with significantly differential regulation between pre- and posthearing stages. Oligos with significant differences in expression between the SOC and the age-matched brain are marked by triangles (green, higher expression in the SOC; red, lower expression in the SOC). The blue horizontal plane depicts the border between developmentally up- and down-regulated oligos. Shown are the mean expression values, normalized to P0 = 1.

FIGURE 7.

Significant enrichment of TAHI in SOC-related gene signatures. A, heat map of 26 TAHI, which are more highly expressed in the SOC than in the age-matched brain at P4 or P25. Oligos were clustered according to the similarity of their normalized expression profiles. Colors indicate the expression level relative to the overall mean intensity (black) of all investigated stages/tissues. Black dots mark statistically significant up-regulation in the SOC (Wilcoxon U test, fc > 2, p < 0.05). B, confirmation of seven up-regulated transcripts in the SOC (cf. A) by semiquantitative RT-PCR (actin as a loading control). Similar results were obtained in at least two independent experiments. C, significant enrichment of oligos associated with hearing impairment in SOC-related genetic programs. Database searches linked 138 oligos of a total of 41,012 (0.34%) to hearing impairment. Of these 138 oligos, 16 (1.75%) were part of the 912 oligos up-regulated in SOC-P4 ↔ Br-P4, 17 (1.06%) were part of the 1,609 oligos up-regulated in the SOC-P25 ↔ Br-P25, and 7 (1.61%) were up-regulated in the SOC at both stages. The enrichment of TAHI was significant for all three SOC samples. **, p < 0.01; ***, p < 0.001.