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Long-distance retinoid signaling in the zebra finch brain.

Roeske TC, Scharff C, Olson CR, Nshdejan A, Mello CV - PLoS ONE (2014)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Psychology, Hunter College, City University of New York, New York, New York, United States of America.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus

ATRA mapping in brain sections through a reporter cell assay.A and B: Schematic overview of ATRA reporter cell assay. A: Reporter cells (they contain a LacZ gene under a retinoic acid response element, or RARE, and express retinoic acid and retinoid X receptors - RAR/RXRs - needed for ATRA induced gene expression) are seeded onto a Petri dish; a freshly cut brain section is placed into the dish and attaches to the cell monolayer. B: ATRA locally generated in the brain slice reaches a reporter cell, binds to RAR/RXR complexes and causes LacZ expression, revealed as blue label by LacZ/X-gal staining. C and D: LacZ expression in reporter cells is specifically induced by ATRA. C (negative control 1): In the absence of ATRA, reporter cells are LacZ-negative and do not turn blue upon LacZ/X-gal staining. D (positive control): Labeling is generated when ATRA is added to the medium. E (negative control 2): A slice co-cultured with an F9 cell line without RARE and LacZ does not generate blue signal upon LacZ/X-gal staining. F: A slice from an adult male bird co-cultured with reporter cells results in blue labeling in regions where ATRA is present. Blue labeling is seen under song nucleus HVC, which expresses zRalDH, but not in cells without overlying tissue (top), or in cells that underlie a part of the tissue that does not contain ATRA (bottom). Photos in C–F were taken through the bottom of the Petri dish. Scale bars: for C, D  = 200µm; for E, F = 100µm.
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pone-0111722-g007: ATRA mapping in brain sections through a reporter cell assay.A and B: Schematic overview of ATRA reporter cell assay. A: Reporter cells (they contain a LacZ gene under a retinoic acid response element, or RARE, and express retinoic acid and retinoid X receptors - RAR/RXRs - needed for ATRA induced gene expression) are seeded onto a Petri dish; a freshly cut brain section is placed into the dish and attaches to the cell monolayer. B: ATRA locally generated in the brain slice reaches a reporter cell, binds to RAR/RXR complexes and causes LacZ expression, revealed as blue label by LacZ/X-gal staining. C and D: LacZ expression in reporter cells is specifically induced by ATRA. C (negative control 1): In the absence of ATRA, reporter cells are LacZ-negative and do not turn blue upon LacZ/X-gal staining. D (positive control): Labeling is generated when ATRA is added to the medium. E (negative control 2): A slice co-cultured with an F9 cell line without RARE and LacZ does not generate blue signal upon LacZ/X-gal staining. F: A slice from an adult male bird co-cultured with reporter cells results in blue labeling in regions where ATRA is present. Blue labeling is seen under song nucleus HVC, which expresses zRalDH, but not in cells without overlying tissue (top), or in cells that underlie a part of the tissue that does not contain ATRA (bottom). Photos in C–F were taken through the bottom of the Petri dish. Scale bars: for C, D  = 200µm; for E, F = 100µm.

Mentions: To localize sites of ATRA presence in brain sections, we used a reporter cell assay (fig. 7.A,B) consisting of a monolayer of mouse F9 cells carrying a LacZ reporter under the control of a retinoic acid response element (RARE). ATRA produced by tissue samples placed on top of the reporter cell layer induces LacZ expression [46], [48]. To detect ATRA-induced gene expression in entire brain sections, we used a modified assay (details in fig. 7.C–E, Methods, and table S2). We observed spatially specific LacZ expression in all birds (fig. 7.F). Reporter cells that were not touched by the co-cultured tissue sections did not show any blue LacZ staining, even if located directly adjacent to the edge of tissue that generated staining in the cell monolayer (fig. 7.F). In addition, boundaries between tissue regions that expressed LacZ and those that did not could be relatively sharp, as was the case for some regions close to HVC (fig. 7.F), indicating that ATRA did not freely diffuse across the reporter cell monolayer, but reflects sites of ATRA presence in the brain sections.


Long-distance retinoid signaling in the zebra finch brain.

Roeske TC, Scharff C, Olson CR, Nshdejan A, Mello CV - PLoS ONE (2014)

ATRA mapping in brain sections through a reporter cell assay.A and B: Schematic overview of ATRA reporter cell assay. A: Reporter cells (they contain a LacZ gene under a retinoic acid response element, or RARE, and express retinoic acid and retinoid X receptors - RAR/RXRs - needed for ATRA induced gene expression) are seeded onto a Petri dish; a freshly cut brain section is placed into the dish and attaches to the cell monolayer. B: ATRA locally generated in the brain slice reaches a reporter cell, binds to RAR/RXR complexes and causes LacZ expression, revealed as blue label by LacZ/X-gal staining. C and D: LacZ expression in reporter cells is specifically induced by ATRA. C (negative control 1): In the absence of ATRA, reporter cells are LacZ-negative and do not turn blue upon LacZ/X-gal staining. D (positive control): Labeling is generated when ATRA is added to the medium. E (negative control 2): A slice co-cultured with an F9 cell line without RARE and LacZ does not generate blue signal upon LacZ/X-gal staining. F: A slice from an adult male bird co-cultured with reporter cells results in blue labeling in regions where ATRA is present. Blue labeling is seen under song nucleus HVC, which expresses zRalDH, but not in cells without overlying tissue (top), or in cells that underlie a part of the tissue that does not contain ATRA (bottom). Photos in C–F were taken through the bottom of the Petri dish. Scale bars: for C, D  = 200µm; for E, F = 100µm.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4230966&req=5

pone-0111722-g007: ATRA mapping in brain sections through a reporter cell assay.A and B: Schematic overview of ATRA reporter cell assay. A: Reporter cells (they contain a LacZ gene under a retinoic acid response element, or RARE, and express retinoic acid and retinoid X receptors - RAR/RXRs - needed for ATRA induced gene expression) are seeded onto a Petri dish; a freshly cut brain section is placed into the dish and attaches to the cell monolayer. B: ATRA locally generated in the brain slice reaches a reporter cell, binds to RAR/RXR complexes and causes LacZ expression, revealed as blue label by LacZ/X-gal staining. C and D: LacZ expression in reporter cells is specifically induced by ATRA. C (negative control 1): In the absence of ATRA, reporter cells are LacZ-negative and do not turn blue upon LacZ/X-gal staining. D (positive control): Labeling is generated when ATRA is added to the medium. E (negative control 2): A slice co-cultured with an F9 cell line without RARE and LacZ does not generate blue signal upon LacZ/X-gal staining. F: A slice from an adult male bird co-cultured with reporter cells results in blue labeling in regions where ATRA is present. Blue labeling is seen under song nucleus HVC, which expresses zRalDH, but not in cells without overlying tissue (top), or in cells that underlie a part of the tissue that does not contain ATRA (bottom). Photos in C–F were taken through the bottom of the Petri dish. Scale bars: for C, D  = 200µm; for E, F = 100µm.
Mentions: To localize sites of ATRA presence in brain sections, we used a reporter cell assay (fig. 7.A,B) consisting of a monolayer of mouse F9 cells carrying a LacZ reporter under the control of a retinoic acid response element (RARE). ATRA produced by tissue samples placed on top of the reporter cell layer induces LacZ expression [46], [48]. To detect ATRA-induced gene expression in entire brain sections, we used a modified assay (details in fig. 7.C–E, Methods, and table S2). We observed spatially specific LacZ expression in all birds (fig. 7.F). Reporter cells that were not touched by the co-cultured tissue sections did not show any blue LacZ staining, even if located directly adjacent to the edge of tissue that generated staining in the cell monolayer (fig. 7.F). In addition, boundaries between tissue regions that expressed LacZ and those that did not could be relatively sharp, as was the case for some regions close to HVC (fig. 7.F), indicating that ATRA did not freely diffuse across the reporter cell monolayer, but reflects sites of ATRA presence in the brain sections.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Psychology, Hunter College, City University of New York, New York, New York, United States of America.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus