Summary Extrinsic signals controlling generation of neocortical neurons during embryonic life have been difficult to identify. In this study we demonstrate that the dorsal forebrain meninges communicate with the adjacent radial glial endfeet and influence cortical development. We took advantage of Foxc1 mutant mice with defects in forebrain meningeal formation. Foxc1 dosage and loss of meninges correlated with a dramatic reduction in both neuron and intermediate progenitor production and elongation of the neuroepithelium. Several types of experiments demonstrate that retinoic acid (RA) is the key component of this secreted activity. In addition, Rdh10 and Raldh2 expressing cells in the dorsal meninges were either reduced or absent in the Foxc1 mutants and Rdh10 mutants had a cortical phenotype similar to the Foxc1-null mutants. Lastly, in utero RA treatment rescued the cortical phenotype in Foxc1 mutants. These results establish RA as a potent, meningeal-derived cue required for successful corticogenesis.

All-trans-retinoic acid (atRA) provides essential support to diverse biological systems and physiological processes. Epithelial differentiation and its relationship to cancer and embryogenesis have typified intense areas of interest into atRA function. Recently, however, interest in atRA action in the nervous system, the immune system, energy balance and obesity has increased considerably, especially concerning postnatal function. atRA action depends on atRA biosynthesis: defects in retinoid-dependent processes increasingly relate to defects in atRA biogenesis. Considerable evidence indicates that physiological atRA biosynthesis occurs via a regulated process, consisting of a complex interaction of retinoid binding-proteins and retinoid recognizing enzymes. An accrual of biochemical, physiological and genetic data have identified specific functional outcomes for the retinol dehydrogenases, RDH1, RDH10, and DHRS9, as physiological catalysts of the first step in atRA biosynthesis, and for the retinal dehydrogenases RALDH1, RALDH2, and RALDH3, as catalysts of the second and irreversible step. Each of these enzymes associates with explicit biological processes mediated by atRA. Redundancy occurs, but seems limited. Cumulative data supports a model of interactions among these enzymes with retinoid binding-proteins, with feedback regulation and/or control by atRA via modulating gene expression of multiple participants. The ratio apo-CRBP1/holo-CRBP1 participates by influencing retinol flux into and out of storage as retinyl esters, thereby modulating substrate to support atRA biosynthesis. atRA biosynthesis requires presence of both an RDH and an RALDH: conversely, absence of one isozyme of either step does not indicate lack of atRA biosynthesis at the site.

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We report a sensitive LC (liquid chromatography)/MS/MS assay using selected reaction monitoring to quantify RA (retinoic acid), which is applicable to biological samples of limited size (10-20 mg of tissue wet weight), requires no sample derivatization, provides mass identification and resolves atRA (all-trans-RA) from its geometric isomers. The assay quantifies over a linear range of 20 fmol to 10 pmol, and has a 10 fmol limit of detection at a signal/noise ratio of 3. Coefficients of variation are: instrumental, 0.5-2.9%; intra-assay, 5.4+/-0.4%; inter-assay 8.9+/-1.0%. An internal standard (all-trans-4,4-dimethyl-RA) improves accuracy by confirming extraction efficiency and revealing handling-induced isomerization. Tissues of 2-4-month-old C57BL/6 male mice had atRA concentrations of 7-9.6 pmol/g and serum atRA of 1.9+/-0.6 pmol/ml (+/-S.E.M.). Tissue 13-cis-RA ranged from 2.9 to 4.2 pmol/g, and serum 13-cis-RA was 1.2+/-0.3 pmol/ml. CRBP (cellular retinol-binding protein)-null mouse liver had atRA approximately 30% lower than wild-type (P<0.05), but kidney, testis, brain and serum atRA were similar to wild-type. atRA in brain areas of 12-month-old female C57BL/6 mice were (+/-S.E.M.): whole brain, 5.4+/-0.4 pmol/g; cerebellum, 10.7+/-0.3 pmol/g; cortex, 2.6+/-0.4 pmol/g; hippocampus, 8.4+/-1.2 pmol/g; striatum, 15.3+/-4.7 pmol/g. These data provide the first analytically robust quantification of atRA in animal brain and in CRBP-null mice. Direct measurements of endogenous RA should have a substantial impact on investigating target tissues of RA, mechanisms of RA action, and the relationship between RA and chronic disease.

Metabolic activation of retinol into the hormone retinoic acid and metabolism of retinoic acid entail essential aspects of retinoid biology that seem interdependent with functions of retinoid binding-proteins. Cellular retinol binding protein and cellular retinoic acid binding protein enjoy widespread expression and, where expressed, their liganded forms represent the major physiological forms of retinol and retinoic acid, respectively. These retinoid binding proteins may protect cells from the amphipathic properties of retinoids and protect the structurally sensitive retinoids from the cellular milieu. Starting from the perspective that the enzymes most likely to metabolize retinoids in vivo might recognize the major forms of retinoids that occur in vivo, several laboratories have produced results that support a model of retinoid metabolism with prominent roles for the cellular retinoid binding proteins. In this model, liganded cellular retinoid binding proteins serve as substrates for the metabolism of some retinoids (retinol, retinoic acid), restricting access to those enzymes that recognize both the binding protein and the retinoid. Other retinoids (3,4-didehydroretinol, 4-oxo-retinoic acid) liganded to binding-proteins have their metabolism arrested. In its unliganded form, at least one retinoid binding protein (cellular retinol binding protein) serves as a retinoid concentration-sensitive modulator of enzymes that catalyze retinol metabolism. This review will describe the model and the intrinsic relationships among retinoid-specific enzymes and retinoid binding proteins.

We report an improved tandem mass spectrometric assay for retinoic acid (RA) applicable to in vitro and in vivo biological samples. This liquid chromatography tandem mass spectrometric (LC/MS/MS) assay for direct RA quantification is the most sensitive to date, with a 62.5 attomol lower limit of detection and a linear range spanning greater than 4 orders of magnitude (from 250 attomol to 10 pmol). This assay resolves all-trans-RA (atRA) from its endogenous geometric isomers, is applicable to samples of limited size (10-20 mg of tissue), and functions with complex biological matrixes. Coefficients of variation are as follows: instrumental, ≤2.6%; intraday, 5.2% ± 0.7%; interday, 6.7% ± 0.9%. In vitro capabilities are demonstrated by quantification of endogenous RA and RA production (from retinol) in primary cultured astrocytes. Quantification of endogenous atRA and its geometric isomers in 129SV mouse serum and tissues (liver, kidney, adipose, muscle, spleen, testis, and brain) reveals in vivo utility of the assay. The ability to discriminate spatial concentrations of RA in vivo is illustrated with C57BL/6 mouse brain loci (hippocampus, cortex, olfactory bulb, thalamus, cerebellum, and striatum), as well as with Lewis rat proximal/distal mammary gland regions during various morphological stages: virgin, early pregnancy (e7), late pregnancy (e20), lactating (day 4), involuting day 1, and involuting day 11. This assay provides the sensitivity necessary for direct, endogenous RA quantification necessary to elucidate RA function, e.g., in neurogenesis, morphogenesis, and the contribution of altered RA homeostasis to diseases, such as Alzheimer's disease, type 2 diabetes, obesity, and cancer.Metabolism activates vitamin A (retinol) into retinoic acid (RA), which controls physiological processes including development, nervous system function, immune response, cell proliferation and differentiation, and reproduction. 1-3 Expression loci of retinoidspecific binding proteins, enzymes, and receptors that contribute to RA generation, signaling, and catabolism indicate that RA concentrations in vivo are temporally/spatially controlled to produce the individual actions of vitamin A. [4][5][6][7][8] Recent investigations have linked alterations in retinoid metabolism to aberrant neurogenesis, aberrant mammary gland morphogenesis, and to diseases, including Alzheimer's disease, type 2 diabetes, obesity, and cancer. 9-14 These studies and others postulate alterations in RA as mechanistically important but have not quantified RA directly. Therefore, analytically rigorous measurements capable of sensitively discriminating changes in endogenous RA levels either Here we report an improved and versatile method for direct quantification of atRA and its isomers in cultured cells, serum, and tissues with increased sensitivity and greater facility to monitor retinoid metabolism in vitro and in vivo. This assay provides the best sensitivity to date for quantifying RA and could be adapted to monitor RA metabolites. EXPERIMENTAL SEC...

Cellular binding-proteins (BP), including CRBP1, CRBP2, CRABP1, CRABP2, and FABP5, shepherd the poorly aqueous soluble retinoids during uptake, metabolism and function. Holo-BP promote efficient use of retinol, a scarce but essential nutrient throughout evolution, by sheltering it and its major metabolite all-trans-retinoic acid from adventitious interactions with the cellular milieu, and by imposing specificity of delivery to enzymes, nuclear receptors and other partners. Apo-BP reflect cellular retinoid status and modify activities of retinoid metabolon enzymes, or exert non-canonical actions. High ligand binding affinities and the nature of ligand sequestration necessitate external factors to prompt retinoid release from holo-BP. One or more of cross-linking, kinetics, and colocalization have identified these factors as RDH, RALDH, CYP26, LRAT, RAR and PPARβ/δ. Michaelis-Menten and other kinetic approaches verify that BP channel retinoids to select enzymes and receptors by protein-protein interactions. Function of the BP and enzymes that constitute the retinoid metabolon depends in part on retinoid exchanges unique to specific pairings. The complexity of these exchanges configure retinol metabolism to meet the diverse functions of all-trans-retinoic acid and its ability to foster contrary outcomes in different cell types, such as inducing apoptosis, differentiation or proliferation. Altered BP expression affects retinoid function, for example, by impairing pancreas development resulting in abnormal glucose and energy metabolism, promoting predisposition to breast cancer, and fostering more severe outcomes in prostate cancer, ovarian adenocarcinoma, and glioblastoma. Yet, the extent of BP interactions with retinoid metabolon enzymes and their impact on retinoid physiology remains incompletely understood.

We report robust HPLC/UV methods for quantifying retinyl esters (RE), retinol (ROL) and retinal (RAL) applicable to diverse biological samples, with lower limits of detection of 0.7 pmol, 0.2 pmol, and 0.2 pmol, respectively, and linear ranges >3 orders of magnitude. These assays function well with small, complex biological samples (10-20 mg tissue). Coefficients of variation range from: intra-day, 5.9-10.0%; inter-day, 5.9-11.0%. Quantification of endogenous RE, ROL, and RAL in mouse serum and tissues (liver, kidney, adipose, muscle, spleen, testis, skin, brain, and brain regions) reveals utility. Ability to discriminate spatial concentrations of ROL and RE is illustrated with C57BL/6 mouse brain loci (hippocampus, cortex, olfactory bulb, thalamus, cerebellum, and striatum.) We also developed a method to distinguish isomeric forms of ROL to investigate precursors of retinoic acid. The ROL isomer assay has limits of detection between 3.5-4.5 pmol and a similar linear range and % CV as the ROL/RE and RAL assays. The assays described here provide for sensitive and rigorous quantification of endogenous RE, ROL, and RAL to elucidate retinoid homeostasis in disease states, such as Alzheimer's disease, type 2 diabetes, obesity, and cancer.