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Vitamin A deficiency studies have been carried out since the early 1900s. Initially, these studies led to the identification of fat soluble A as a unique and essential component of the diet of rodents, birds, and humans. Continuing work established that vitamin A deficiency produces biochemical and physiological dysfunction in almost every vertebrate organ system from conception to death. This chapter begins with a review of representative historical and current studies that used the nutritional vitamin A deficiency research model to gain an understanding of the many roles vitamin A plays in prenatal and postnatal development and well-being. This is followed by a discussion of recent studies that show specific effects of vitamin A deficiency on prenatal development and postnatal maintenance of the olfactory epithelium, brain, and heart. Vitamin A deficiency studies have helped define the necessity of vitamin A for the health of all vertebrates, including farm animals, but the breadth of deficient states and their individual effects on health have not been fully determined. Future work is needed to develop tools to assess the complete vitamin A status of an organism and to define the levels of vitamin A that optimally support molecular and systems level processes during all ages and stages of life.

Embryonic development is controlled by retinoids, and one approach that has been used to investigate the mechanisms for retinoid actions in developmental processes has been to study the effects of adding retinoids to cultures of pluripotent embryonic stem cells (ESC). To date, most in vitro retinoid research has been directed at deciphering the actions of all-trans retinoic acid (atRA). atRA is a derivative of all-trans retinol (a.k.a. vitamin A, VA), which mammals can generate via an enzyme-catalyzed pathway. atRA's effects on development result from its (1) activation of receptor complexes (RARs and RXRs) in the nucleus which then bind to and activate RA response elements (RAREs) in genes and (2) its interactions with processes that are initiated in the cytoplasm. While much work has focused on the impact of atRA on cell differentiation, VA, itself, has been shown to exert effects on the maintenance of ESC identity that are not dependent upon classic RA-signaling pathways. In this chapter, we present results from our laboratory and others using well-documented approaches for investigating the effects of retinoids on the differentiation of ESC in vitro and introduce a novel method that uses chemically-defined growth conditions. The merits of each approach are discussed.

To determine whether hepatic depletion of vitamin A (VA) stores has an effect on the postnatal heart, studies were carried out with mice lacking liver retinyl ester stores fed either a VA-sufficient (LRVAS) or VA-deficient (LRVAD) diet (to deplete circulating retinol and extrahepatic stores of retinyl esters). There were no observable differences in the weights or gross morphology of hearts from LRVAS or LRVAD mice relative to sex-matched, age-matched, and genetically matched wild-type (WT) controls fed the VAS diet (WTVAS), but changes in the transcription of functionally relevant genes were consistent with a state of VAD in LRVAS and LRVAD ventricles. In silico analysis revealed that 58/67 differentially expressed transcripts identified in a microarray screen are products of genes that have DNA retinoic acid response elements. Flow cytometric analysis revealed a significant and cell-specific increase in the number of proliferating Sca-1 cardiac progenitor cells in LRVAS animals relative to WTVAS controls. Before myocardial infarction, LRVAS and WTVAS mice had similar cardiac systolic function and structure, as measured by echocardiography, but, unexpectedly, repeat echocardiography demonstrated that LRVAS mice had less adverse remodeling by 1 wk after myocardial infarction. Overall, the results demonstrate that the adult heart is responsive to retinoids, and, most notably, reducing hepatic VA stores (while maintaining circulating levels of VA) impacts ventricular gene expression profiles, progenitor cell numbers, and response to injury.

Pluripotent mouse embryonal carcinoma (mEC) and mouse embryonic stem (mES) cells differentiate into several cell lineages upon retinoic acid (RA) addition. Differentiation is facilitated, in part, by RA activation of nuclear RA receptors (RARs) that bind to DNA response elements located in the promoters of target genes. The purpose of the studies reported here was to immunolocalize RARα and RARγ protein in mEC and mES cells and in their RA-induced differentiated progeny. Fixed cells were reacted with three different RARα antibodies and one RARγ antibody. Pluripotent and differentiated mEC and mES cells showed positive nuclear immunoreactivity with all antibodies tested. Two RARα antibodies also showed positive reactivity in the cytoplasm. Surprisingly, our results revealed variability in immunofluorescence intensity and in RARα and RARγ distribution from one cell to the other, suggesting that RARα and RARγ protein levels were not synchronous throughout the cell population. The results indicate that RARα and RARγ are present in pluripotent and differentiating mEC and mES cells and suggest that the expression of these proteins is dynamic.

Vitamin A (VA), all-trans-retinol (at-ROL), and its derivative, all-trans-retinoic acid (at-RA), are required for neuron development. The effects of these retinoids are dependent upon the nutritional status of the rat and tissue-specific dynamics of retinoid access and utilization. The purpose of this study was to determine the status of at-ROL and at-RA in the peripheral olfactory organ of postnatal rats fed a normal diet and rats fed a VA-deficient (VAD) diet. Extracted retinoids were analyzed by HPLC. Resolved sample peaks were identified by comparing their elution times and spectra with those of authentic standards. Mean at-RA and at-ROL concentrations of 23 pmol/g olfactory tissue and 0.13 nmol/g, respectively, were recovered from olfactory tissue. The ratio of at-RA:at-ROL in olfactory was approximately 2 times that in testis and 200 times that in liver. at-ROL was depleted from the liver and olfactory organ of rats fed a VAD diet from birth to 70 d of age. Surprisingly, at-RA was still present in olfactory tissue from these rats. At 90 d of age, the VAD rats were frankly deficient and at-RA was no longer detectable in olfactory tissue. The comparatively high ratio of at-RA:at-ROL in the peripheral olfactory organ and the persistence of at-RA in at-ROL-depleted tissues strongly suggests that maintenance of local stores of at-RA is functionally relevant in this tissue.

Vitamin A (VA) is the chemical precursor of retinoic acid (RA), which is critical for embryonic development and for growth, immunity, metabolism, and cell differentiation in postnatal regenerating systems such as skin, sensory organs, and stem cell niches in the brain. VA is also essential for embryonic heart development, and we hypothesized that Vitamin A might exert an effect on the postnatal heart similar to what is observed for other tissues. Here, we report the global transcriptional profiles of wild-type (WT) mice fed a VA sufficient diet (VAS) compared with retinyl acyl transferase (LRAT) knock-out mice fed either a VAS or VA deficient (VAD) diet. Knockout of the LRAT gene alone was sufficient to induce differential expression of 576 genes relative to WT. Feeding LRAT mutant mice a VAD diet resulted in a change in the relative expression levels of 257 genes relative to LRAT mutant mice fed a VAS diet. As expected, we observed transcriptional alterations related to Vitamin A metabolism, including an increase in the gene encoding cellular retinoid binding protein 7 and down-regulation of the retinol metabolic enzymes Cy1a2 and Cyp2a4. Importantly, several cardiac genes not previously known to require VA were perturbed, including the gene encoding B-type natriuretic peptide, which was down-regulated in mutant mice irrespective of diet, and A-type natriuretic peptide, which was decreased only in mice fed the VAD diet. There was also a striking effect of VAD on genes important for immune responses, which could have an impact on the wound healing process subsequent to injury of the heart. This is consistent with recent evidence that showed that Vitamin A deficiency influences post-infarct ventricular remodeling in rats. In summary, this is the first microarray study of Vitamin A deficiency in the postnatal heart, which suggests mechanisms by which Vitamin A depletion may alter myocardial maintenance and repair after injury.

It has been reported that retinoids, such as retinoic acid (RA) and retinol (ROL), dissolved in aqueous solutions are susceptible to oxidative damage when exposed to light, air, and relatively high temperatures, conditions that are normal for culturing stem cells. Thus, questions arise regarding the interpretation of results obtained from studies of mouse embryonic stem cells exposed to retinoids because their isomerization state, their stability in culture conditions, and their interactions with other potential differentiation factors in growth media could influence developmental processes under study. Media samples were supplemented with retinoids and exposed to cell culture conditions with and without mouse embryonic stem cells (mESC), and retinoids were extracted and analyzed using HPLC. To determine whether retinoids are stable in media supplemented with fetal bovine serum (FBS) or in chemically-defined, serum-free media, mESC adapted to each type of growth media were investigated. Studies reported here indicate there was little loss or isomerization of at-RA, 9-cis-RA, 13-cis-RA, or ROL in cell cultures grown in serum-supplemented media when cell cultures were maintained in the dark and manipulated and observed under yellow light. In contrast, the stability of both at-RA and ROL were determined to be greatly reduced in serum-free media as compared with serum-supplemented media. Addition of 6 mg/ml bovine serum albumin was found to stabilize retinoids in serum-free media. It was also determined that ROL is less stable than RA in cell culture conditions.

Work from our laboratory suggests that retinoic acid (RA) influences neuron development in the postnatal olfactory epithelium (OE). The studies reported here were carried out to identify and localize retinaldehyde dehydrogenase (RALDH) expression in postnatal rat OE to gain a better understanding of potential in vivo RA synthesis sites in this continuously regenerating tissue. RALDH 1, 2, and 3 mRNAs were detected in postnatal rat olfactory tissue by RT-PCR analysis, but RALDH 1 and 2 transcripts were predominant. RALDH 1 immunoreactivity was localized to sustentacular cells in the OE and to Bowman's gland cells, and GFAP+/p75− olfactory ensheathing cells (OECs) in the underlying lamina propria (LP). RALDH 2 did not colocalize with RALDH 1, but appeared to be expressed in GFAP−/RALDH 1− OECs as well as in unidentified structures in the LP. Cellular RA binding protein (CRABP II) colocalized with RALDH 1. Cellular retinol/retinaldehyde binding protein (CRBP I) was localized to RALDH 1+ sites in the OE and LP and RALDH 2+ sites, primarily surrounding nerve fiber bundles in the LP. Vitamin A deficiency altered RALDH 1, but not RALDH 2 protein expression. The isozymes and binding proteins exhibited random variability in levels and areas of expression both within and between animals. These findings support the hypothesis that RA is synthesized in the postnatal OE (catalyzed by RALDH 1) and underlying LP (differentially catalyzed by RALDH 1 and RALDH 2) at sites that could influence the development, maturation, targeting, and/or turnover of olfactory receptor neurons throughout the olfactory organ. J. Comp. Neurol. 496:149–171, 2006. © 2006 Wiley-Liss, Inc.

Cellular retinoic acid binding proteins, types I and II (CRABP I and II), are cytosolic proteins that exhibit a binding preference for all-trans retinoic acid. As part of a larger study to determine whether retinoic acid plays a role in neurogenesis in vivo, we questioned whether CRABP II is present in rat postnatal olfactory epithelium (OE), a sensory tissue that continually replaces neurons throughout adult life. We have determined that both CRABP II and CRABP I proteins and the mRNAs that encode them are present in postnatal rat OE. Immunoreactivity with CRABP II and CRABP I antibodies was not observed in the nasal respiratory epithelium. Double immunolabeling experiments, conducted with antibodies showing specificity for each antigen, indicate that CRABP II and CRABP I are found in different cell types within the olfactory neuroepithelium. We also asked whether CRABP II is expressed in the postnatal rat retina, a neural tissue that is not known to show neuron replacement during adult life. CRABP type II immunoreactivity was not observed in the mature rat retina. The presence of CRABP II in postnatal OE and its absence from mature retina is consistent with previous reports indicating that the distribution of CRABP II in adult mammals is restricted to tissue systems that exhibit ongoing growth and differentiation throughout life.