Transient retinoic acid signaling confers anterior-posterior polarity to the inner ear.

Vertebrate hearing and balance are based in complex asymmetries of inner ear structure. Here, we identify retinoic acid (RA) as an extrinsic signal that acts directly on the ear rudiment to affect its compartmentalization along the anterior-posterior axis. A rostrocaudal wave of RA activity, generated by tissues surrounding the nascent ear, induces distinct responses from anterior and posterior halves of the inner ear rudiment. Prolonged response to RA by posterior otic tissue correlates with Tbx1 transcription and formation of mostly nonsensory inner ear structures. By contrast, anterior otic tissue displays only a brief response to RA and forms neuronal elements and most sensory structures of the inner ear.

Retinoic acid influences neuronal migration from the ganglionic eminence to the cerebral cortex.

The ganglionic eminence contributes cells to several forebrain structures including the cerebral cortex, for which it provides GABAergic interneurons. Migration of neuronal precursors from the retinoic-acid rich embryonic ganglionic eminence to the cerebral cortex is known to be regulated by several factors, but retinoic acid has not been previously implicated. We found retinoic acid to potently inhibit cell migration in slice preparations of embryonic mouse forebrains, which was reversed by an antagonist of the dopamine-D(2) receptor, whose gene is transcriptionally regulated by retinoic acid. Histone-deacetylase inhibitors, which amplify nuclear receptor-mediated transcription, potentiated the inhibitory effect of retinoic acid. Surprisingly, when retinoic acid signalling was completely blocked with a pan-retinoic acid receptor antagonist, this also decreased cell migration into the cortex, implying that a minimal level of endogenous retinoic acid is necessary for tangential migration. Given these opposing effects of retinoic acid in vitro, the in vivo contribution of retinoic acid to migration was tested by counting GABAergic interneurons in cortices of adult mice with experimental reductions in retinoic acid signalling: a range of perturbations resulted in significant reductions in the numerical density of some GABAergic interneuron subpopulations. These observations suggest functions of retinoic acid in interneuron diversity and organization of cortical excitatory-inhibitory balance.

Transcriptomics of Gabra4 knockout mice reveals common NMDAR pathways underlying autism, memory, and epilepsy.

Autism spectrum disorder (ASD) is a neuronal developmental disorder with impaired social interaction and communication, often with abnormal intelligence and comorbidity with epilepsy. Disturbances in synaptic transmission, including the GABAergic, glutamatergic, and serotonergic systems, are known to be involved in the pathogenesis of this disorder, yet we do not know if there is a common molecular mechanism. As mutations in the GABAergic receptor subunit gene GABRA4 are reported in patients with ASD, we eliminated the Gabra4 gene in mice and found that the Gabra4 knockout mice showed autistic-like behavior, enhanced spatial memory, and attenuated susceptibility to pentylenetetrazol-induced seizures, a constellation of symptoms resembling human high-functioning autism. To search for potential molecular pathways involved in these phenotypes, we performed a hippocampal transcriptome profiling, constructed a hippocampal interactome network, and revealed an upregulation of the NMDAR system at the center of the converged pathways underlying high-functioning autism-like and anti-epilepsy phenotypes.

Integrating retinoic acid signaling with brain function.

The vitamin A derivative retinoic acid (RA) regulates the transcription of about a 6th of the human genome. Compelling evidence indicates a role of RA in cognitive activities, but its integration with the molecular mechanisms of higher brain functions is not known. Here we describe the properties of RA signaling in the mouse, which point to unknown means through which RA actions are modified and reinforced at selected brain sites. The locations of RA signaling for the developing dorsal forebrain undergo slow, gradual changes over the life cycle except for two brief periods of accelerated shifts, which coincide with periods of enhanced developmental vulnerability. In the functional cerebral cortex, RA signaling delineates regions with immature, plastic neuronal characteristics, within which the expression of hundreds of genes is differentially regulated. Many of these are involved in neuronal ligand-receptor interactions and signaling cascades for activity dependent gene expression. We propose that RA functions in the brain by contributing topographical information and life cycle changes to combinatorial transcriptional mechanisms and that in the postnatal cortex RA signaling designates domains of modifiable neuronal circuitry.

Retinoic acid signaling in the functioning brain.

Retinoic acid, an active form of vitamin A, regulates gene expression throughout the body, and many components of the signaling system through which it acts are present in the brain. Very little is known, however, about how retinoic acid functions in neurobiological systems. Several studies have provided evidence that retinoic acid plays a role in sleep, learning, and memory, but the precise mechanisms through which it influences these processes remain unclear. All of these processes involve local or long-range inhibition and synchronized neuronal activity between separate locations in the brain. A critical component in the generation of the synchronized firing of cortical neurons (cortical synchrony) is a network of inhibitory interneurons containing parvalbumin, a cell population affected by retinoid perturbations, such as exposure to a vitamin A overdose. An understanding of the role of retinoids in normal brain function would provide clues to the long-standing question of whether abnormalities in retinoic acid signaling contribute to the pathogenesis of some brain diseases with uncertain etiologies that involve both genetic and environmental factors.

A retinoic-acid critical period in the early postnatal mouse brain.

BACKGROUND: A normal supply of vitamin A, which regulates gene expression through its active form retinoic acid, is required by many organs; both excess and deficiency can be teratogenic. Very little is known about the role of retinoic acid in maturation of the mammalian forebrain. METHODS: As retinoic acid cannot be visualized directly, we mapped its actions in the forebrain with indirect morphologic methods and by applying retinoic acid overdoses to early postnatal mice. RESULTS: During this time, the morphologic indicators of retinoic acid actions are localized mainly in the limbic system and they undergo rapid changes. Retinoic acid overdoses can cause lasting behavioral abnormalities that point to disrupted limbic functions. In the anterior cingulate cortex, inhibitory interneurons are affected, and in the hippocampus, primarily the dentate gyrus is abnormal. CONCLUSIONS: Retinoic acid is involved in functional maturation of limbic regions of the forebrain with a critical stage early postnatally in mice, when their brains are particularly vulnerable to vitamin A perturbations. This developmental time in mice compares with the second trimester of gestation in humans, a stage when in genetically predisposed individuals the corresponding brain regions are known to pass through a period of increased susceptibility to environmental disturbances.

Retinoic acid signaling in the brain marks formation of optic projections, maturation of the dorsal telencephalon, and function of limbic sites.

As retinoic acid (RA) is known to regulate the expression of many neuronal proteins, it is likely to influence overall development and function of the brain; few particulars, however, are available about its role in neurobiological contexts due mainly to problems in RA detection. To ask whether the function of RA in the rostral brain is concentrated in particular neurobiological systems, we compared sites of RA synthesis and actions, as detected by RA signaling in reporter mice, for embryonic and adult ages. We found that most sites of RA actions in the forebrain do not colocalize with RA synthesis, consistent with a dominant RA supply by diffusion and the circulation. The changing RA patterns distinguish preferentially two complex functional schemes. (1) Within the visual system when the first optic axons grow toward their targets, RA signaling delineates the topographical adjustment of the retinal map, which is encoded in the coordinates of the visual world, to central visual maps, which are formed in the segmental brain coordinates. (2) The second scheme begins early in forebrain morphogenesis as a distinction of the dorsal telencephalon. With progressing development, and in the adult, the RA patterns then focus on widely distributed structures, most of which belong to the limbic system. These are sites in which emotional perception is combined with higher cognitive processes and in which normal function requires ongoing remodeling of synaptic connections, indicating that the developmental role of RA in promotion of neuronal differentiation programs continues in the adult brain for highly flexible neural circuits. J. Comp. Neurol. 470:297-316, 2004.

Differential expression of components of the retinoic acid signaling pathway in the adult mouse olfactory epithelium.

Position within a tissue often correlates with cellular phenotype, for example, differential expression of odorant receptors and cell adhesion molecules across the olfactory mucosa (OM). The association between position and phenotype is often paralleled by gradations in the concentration of retinoic acid (RA), caused by differential expression of the RA synthetic enzymes, the retinaldehyde dehydrogenases (RALDH). We show here that RALDH-1, -2, and -3 are enriched in the sustentacular cells, deep fibroblasts of the lamina propria, and the superficial fibroblasts, respectively, of the ventral and lateral OM as compared to the dorsomedial OM. The shift from high to low expression of the RALDHs matches the boundary defined by the differential expression of OCAM/mamFasII. Further, we found that RA-binding proteins are expressed in the epithelium overlying the RALDH-3 expressing fibroblasts of the lamina propria. Both findings suggest that local alterations in RA concentration may be more important than a gradient of RA across the epithelial plane, per se. In addition, RALDH-3 is found in a small population of basal cells in the ventral and lateral epithelium, which expand and contribute to the neuronal lineage following MeBr lesion. Indeed, transduction with a retrovirus expressing a dominant negative form of retinoic acid receptor type alpha blocks the reappearance of mature, olfactory marker protein (OMP) (+) olfactory neurons as compared to empty vector. These results support the notion of a potential role for RA, both in maintaining the spatial organization of the normal olfactory epithelium and in reestablishing the neuronal population during regeneration after injury.

Detection of retinoic acid catabolism with reporter systems and by in situ hybridization for CYP26 enzymes.

Retinoic acid (RA), an active form of vitamin A, is essential for life in vertebrates, owing to its capacity of influencing expression of a sizable fraction of all genes and proteins. It functions via two modes: (1) as controlling ligand for specific transcription factors in the nucleus it stimulates or inhibits gene expression from RA response elements in gene promoters; (2) in non-genomic pathways it activates kinase-signaling cascades that converge with additional influences to regulate gene expression and mRNA translation. RA performs a critical role in morphogenesis of the developing embryo, which is reflected in spatio-temporally changing expression patterns of RA-synthesizing and RA-degrading enzymes and in its biophysical characteristics as a small diffusible lipid. Because its histological localization cannot be directly visualized for technical reasons, its sites of action in vivo are inferred from the locations of the metabolic enzymes and through use of two kinds of RA reporter systems. Here we explain techniques for use of RA reporter cells and RA reporter mice, and we describe in situ hybridization methods for the three major RA-degrading enzymes: CYP26A1, CYP26B1, and CYP26C1. Comparisons of the different indicators for sites of RA signaling demonstrate that local RA peaks and troughs are important for inferring some but not all locations of RA actions. When integrated within cells of living mice, expression of the RA reporter construct is rarely a simple measure of local RA levels, especially in the developing brain, but it appears to provide cues to an RA involvement in site-specific regulatory networks in combination with other spatial determinants.

Retinoids, eye development, and maturation of visual function.

Vitamin A is known to be critical for the beginning of eye development as well as for photoreception in the functional retina. Hardly anything, however, is known about whether retinoic acid (RA)-regulated gene expression also plays a role in the long intervening period, during which the neurobiological retinal structure takes shape. The eye contains a highly intricate architecture of RA-synthesizing (RALDH) and degrading (CYP26) enzymes. Whereas the RALDHs are integrated in the early molecular mechanisms through which the dorso-ventral retina organization is established, the CYP26 enzymes are not necessary for this process and no molecular targets that match their retinal expression pattern have yet been identified. In this article we describe that CYP26 expression in the mouse is most distinctive during later stages of retina formation. Throughout development CYP26A1 degrades RA in a horizontal region that extends across the retina, but during later embryonic and postnatal retina maturation this function is reinforced by another enzyme, CYP26C1. RA applications at this stage do not affect the RALDHs but cause differential changes in CYP26 expression: Cyp26a1 is up-regulated, but more rapidly by 9-cis than all-trans RA, Cyp26c1 is down-regulated, and Cyp26b1, which is undetectable in the normal mouse retina, is strongly activated in retinal ganglion cells. The dynamic regulation in RA-difference patterns by the CYP26 enzymes may set up spatial constellations for expression of genes involved in formation of retinal specializations for higher acuity vision, which are known to form over a prolonged period late in retina development.

Retinoic acid delineates the topography of neuronal plasticity in postnatal cerebral cortex.

Retinoic acid is well recognized to promote neuronal differentiation in the embryonic nervous system, but how it influences the postnatal cerebral cortex remains largely unknown. The domain of highest retinoic acid actions in the cortex of the mouse constricts postnatally to a narrow band that includes the dorsal visual stream and the attentional and executive networks. This band of cortex, which is distinguished by the retinoic acid-synthesizing enzyme RALDH3, exhibits signs of delayed maturation and enhanced plasticity compared to the surrounding cortex, as indicated by suppression of parvalbumin, neurofilament, cytochrome oxidase and perineuronal net maturation, and persistence of the embryonic, polysialated form of the neural cell-adhesion molecule PSA-NCAM. During the first postnatal week, the RALDH3-expressing territory translocates in the caudal cortex from the medial limbic lobe to the adjacent neocortex. This topographical shift requires the neurotrophin NT-3 because in mice lacking neuronal NT-3 the RALDH3 enzyme maintains its early postnatal pattern up to adulthood. In the NT-3-null mutants, expression of the markers, whose topography colocalizes with RALDH3 in the normal cortex, matches the abnormal RALDH3 pattern. This indicates that the uneven retinoic acid distribution serves a role in patterning the maturation and to some extent function of the normal postnatal cerebral cortex.

Retinoic acid synthesis in the postnatal mouse brain marks distinct developmental stages and functional systems.

Retinoic acid (RA) affects development and function of the brain, but little is known about how much is made locally and where it is distributed. To identify RA-sensitive neural processes, we mapped the RA-synthesizing retinaldehyde dehydrogenases (RALDHs) during postnatal brain formation of the mouse. High and stable RALDH expressions mark the basal ganglia, olfactory bulbs, hippocampus and auditory afferents as major sites of RA actions in the functional brain. During the early postnatal period, transient and very high RALDH3 expressions distinguish two developmental events: (i) the colonization of the nucleus accumbens and the olfactory bulbs by neuronal precursors and (ii) the maturation of selected parts of the cerebral cortex. In the cortex, RALDH3 is transiently activated in postmigratory layer II/III neurons during formation of their dendritic arbors and it is transported in their axons across the corpus callosum. RALDH3-expressing cortical regions include most of the limbic lobe, with strongest expression in the anterior cingulate cortex, medial and lateral secondary visual cortices, auditory cortical areas, the secondary motor cortex and some association areas. The transient cortical expression points to a brief RA-critical period during differentiation of the cortical network that serves in the coordination of sensory-motor activity with emotional and recently learned information.

CYP26A1 and CYP26C1 cooperate in degrading retinoic acid within the equatorial retina during later eye development.

In the embryonic mouse retina, retinoic acid (RA) is unevenly distributed along the dorsoventral axis: RA-rich zones in dorsal and ventral retina are separated by a horizontal RA-poor stripe that contains the RA-inactivating enzyme CYP26A1. To explore the developmental role of this arrangement, we studied formation of the retina and its projections in Cyp26a1 null-mutant mice. Expression of several dorsoventral markers was not affected, indicating that CYP26A1 is not required for establishing the dorsoventral retina axis. Analysis of the mutation on a RA-reporter mouse background confirmed, as expected, that the RA-poor stripe was missing in the retina and its projections at the time when the optic axons first grow over the diencephalon. A day later, however, a gap appeared both in retina and retinofugal projections. As explanation, we found that CYP26C1, another RA-degrading enzyme, had emerged centrally in a narrower domain within the RA-poor stripe. While RA applications increased retinal Cyp26a1 expression, they slightly reduced Cyp26c1. These observations indicate that the two enzymes function independently. The safeguard of the RA-poor stripe by two distinct enzymes during later development points to a role in maturation of a significant functional feature like an area of higher visual acuity that develops at its location.

Retinoic acid in the anteroposterior patterning of the zebrafish trunk.

Retinoic acid has been linked to pattern formation in the vertebrate anteroposterior axis. This report describes the spatial and temporal distributions of both endogenous retinoic acid and retinoic acid synthase activity along the anteroposterior axis of neurulating zebrafish embryos, as detected by a transient transgenic assay and by a zymography bioassay. Both retinoic acid levels and synthase activity were found to be highest in anterior regions of the trunk at all of the stages which were analysed. The drug disulfiram inhibited retinoic acid synthase activity in the zebrafish trunk both in vitro and in vivo, and reduced retinoic acid levels in vivo. Disulfiram treatment of neurulating embryos resulted in larvae with hypertrophic wavy notochords, shortened spinal cords and deformed pectoral fins. The results support the hypothesis that retinoic acid plays a role in the coordination of axial patterning at the developing node/zone of involution, as well as in the subsequent development of anterior trunk structures such as the fins.