Long-Distance Retinoid Signaling in the Zebra Finch Brain

All-trans retinoic acid (ATRA), the main active metabolite of vitamin A, is a powerful signaling molecule that regulates large-scale morphogenetic processes during vertebrate embryonic development, but is also involved post-natally in regulating neural plasticity and cognition. In songbirds, it plays an important role in the maturation of learned song. The distribution of the ATRA-synthesizing enzyme, zRalDH, and of ATRA receptors (RARs) have been described, but information on the distribution of other components of the retinoid signaling pathway is still lacking. To address this gap, we have determined the expression patterns of two obligatory RAR co-receptors, the retinoid X receptors (RXR) α and γ, and of the three ATRA-degrading cytochromes CYP26A1, CYP26B1, and CYP26C1. We have also studied the distribution of zRalDH protein using immunohistochemistry, and generated a refined map of ATRA localization, using a modified reporter cell assay to examine entire brain sections. Our results show that (1) ATRA is more broadly distributed in the brain than previously predicted by the spatially restricted distribution of zRalDH transcripts. This could be due to long-range transport of zRalDH enzyme between different nuclei of the song system: Experimental lesions of putative zRalDH peptide source regions diminish ATRA-induced transcription in target regions. (2) Four telencephalic song nuclei express different and specific subsets of retinoid-related receptors and could be targets of retinoid regulation; in the case of the lateral magnocellular nucleus of the anterior nidopallium (lMAN), receptor expression is dynamically regulated in a circadian and age-dependent manner. (3) High-order auditory areas exhibit a complex distribution of transcripts representing ATRA synthesizing and degrading enzymes and could also be a target of retinoid signaling. Together, our survey across multiple connected song nuclei and auditory brain regions underscores the prominent role of retinoid signaling in modulating the circuitry that underlies the acquisition and production of learned vocalizations

RXRα expression in Area X, lMAN, and RA in relation to time of day and age.

<p>A: RXRα expression across the day. Bars represent the difference in normalized expression strength between a song nucleus and the surrounding area (“Δ RXRα inside nucleus-outside”, mean ± SEM); a 0 value indicates no expression difference, positive values indicate more expression inside than outside, negative values the opposite. At different times, expression compared to the surrounds varied significantly in lMAN, but not in Area X or RA; asterisk indicates significance by MANCOVA (Area X: F = 0.79, p = 0.471; lMAN: F = 5.7, p = 0.014; RA: F = 0.31, p = 0.736); for details, see Methods. During the day, the RXRα expression inside and outside lMAN was similar, whereas RXRα expression was lower in lMAN than in the surrounding nidopallium in the morning and evening. B: Representative bright field images of sagittal sections showing variation in RXRα expression in lMAN compared to surrounding nidopallium at different times of the day. C: RXRα expression across ages. Plotted is the same measure of expression strength as in A vs. age (post-hatch days), and linear fits; a significant decrease with age was seen in lMAN (MANCOVA, F = 4.72, p = 0.046) but not in Area X (F = 2.13, p = 0.165) or RA (F = 0.54, p = 0.476).</p

zRalDH protein expression in areas of adult male zebra finch brain that express or lack zRalDH mRNA.

<p>A: Drawing indicates location of images shown in other panels. B: zRalDH mRNA expression by <i>in situ</i> hybridization, level similar to A. C–G: zRalDH protein detection through immunohistochemistry. C: HVC and RA, as well as the fiber tracts extending from HVC to RA (arrows) are labeled. D: lMAN and surrounding nidopallium, as well as Area X, are labeled; labeling in Area X is diffuse and not in somata. Thus, besides HVC and lMAN, which express zRalDH mRNA, protein is present in two song nuclei (X and RA) that lack zRalDH transcript (white arrows in B). E: Detail view of zRalDH protein in RA; labeling is diffuse and not in somata. F: Detail view of zRalDH protein in HVC, somata are labeled. G: High power view of zRalDH in lMAN, somata are labeled. In all panels, frontal is to the right, and dorsal is up. For abbreviations, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111722#pone-0111722-t001" target="_blank">table 1</a>.Scale bars for C = 0.5 mm; D–F = 100µm, G = 50µm.</p

Abbreviations for zebra finch brain structures.

<p>Abbreviations for zebra finch brain structures.</p

RXRγ expression in Area X of adult male zebra finch.

<p>A: Drawing of a parasagittal section of adult brain, indicating detail area shown in B; anterior is to the right, dorsal is up. For abbreviations see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111722#pone-0111722-t001" target="_blank">table 1</a>. B: Detail view of Area X and surrounding area in section processed for RXRγ ISH showing sparse labeled cells in Area X. D: High-magnification view of Area X; black and white arrows depict labeled and unlabeled cells, respectively. Scale bars: 2mm in B, 200µm in C, 50µm in D.</p

CYP26B1 and zRalDH exhibit graded expression in higher auditory areas of adult zebra finch.

<p>The drawing in the top left indicates approximate regions shown in A–E; the drawings on the left depict brain areas shown on the right. The middle and right columns show sections processed for ISH for CYP26B1 and zRalDH. For all images, anterior is to the right and dorsal is up; medial to lateral is represented from top to bottom. CYP26B1 is expressed in a dorsoventral gradient-like pattern throughout the caudal, caudo-medial and caudo-lateral mesopallium (CM, CMM and CLM; arrows in middle panels point to region of high expression). This distribution does not overlap with zRalDH mRNA expression, which is absent in the mesopallium; some cells in the rostral part of the caudal nidopallium express zRalDH (arrows in right panels), tapering off caudally. For other abbreviations, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111722#pone-0111722-t001" target="_blank">table 1</a>. Scale bar  = 0.5 mm.</p

Neural substrates for singing and song learning.

<p>A and B: schematics of the song control system and relevant auditory structures. Represented are the posterior vocal-motor pathway (VMP, blue), and the anterior forebrain pathway (AFP, red). HVC is the origin of both pathways and the entry site of inputs from auditory areas (yellow) into the song system. For abbreviations, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111722#pone-0111722-t001" target="_blank">table 1</a>. In A, brain topography is preserved, for easier comparison with experimental brain sections. In B, indication of broad brain subdivisions (on the right) facilitates comparison with mammalian brains.</p

In the absence of axonal input from HVC, ATRA induced reporter staining is reduced in RA of adult male zebra finches.

<p>Experimental design and time course are indicated by diagrams on the left and on top of photomicrographs. After surgical procedure, birds were allowed to survive for 14 days before reporter assay was performed. A–D: High power views of reporter expression induced in the monolayer by RA; dashed circles indicate position of RA. A and C: control hemispheres with intact HVC-to-RA projections. B and D: experimental hemispheres with HVC lesion (B) or knife-cut fibers (D).</p

Overlapping expression of RARs and RXRs differ across song control nuclei, as well as between most song nuclei and their surroundings.

<p>Expression strength of all receptors based on visual estimates are schematically represented by symbol size (for RARs, data are from Jeong et al. (2005)). For all song nuclei except HVC, the receptor expression profile differs from the surroundings. Thus, each song nucleus has a unique combination of RAR(s)/RXR(s), which may lead to nucleus-specific sets of ATRA effects.</p

RXRα expression in song control nuclei of adult male zebra finch.

<p>A: Schematic of a parasagittal section at the level of song nuclei HVC, RA, Area X, and lMAN. The dashed rectangles indicate the areas shown in B, D, and G. Photos in B–H are bright field views of parasagittal sections hybridized with digoxigenin-labeled RXRα antisense probe. Anterior is right, dorsal is up in all panels. B, C: Expression in RA. Many cells were strongly labeled; the surrounding arcopallium showed less labeling. D-F: Expression in lMAN and Area X. Expression is comparable to the surrounding areas, but lower in Area X/striatum than in lMAN/nidopallium. G, H: Expression in HVC. The high expression is comparable to the adjacent nidopallium. In C, E, F, and H, black arrows depict strongly labeled cells, gray arrows weakly labeled cells, and white arrows unlabeled cells. Scale bars  = 200µm in B, D, G; 20µm in C, E, F, H.</p