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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.

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.

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.

The discovery of retinoic acid receptors arose from research into how vitamins are essential for life. Early studies indicated that Vitamin A was metabolized into an active factor, retinoic acid (RA), which regulates RNA and protein expression in cells. Each step forward in our understanding of retinoic acid in human health was accomplished by the development and application of new technologies. Development cDNA cloning techniques and discovery of nuclear receptors for steroid hormones provided the basis for identification of two classes of retinoic acid receptors, RARs and RXRs, each of which has three isoforms, α, β and ɣ. DNA manipulation and crystallographic studies revealed that the receptors contain discrete functional domains responsible for binding to DNA, ligands and cofactors. Ligand binding was shown to induce conformational changes in the receptors that cause release of corepressors and recruitment of coactivators to create functional complexes that are bound to consensus promoter DNA sequences called retinoic acid response elements (RAREs) and that cause opening of chromatin and transcription of adjacent genes. Homologous recombination technology allowed the development of mice lacking expression of retinoic acid receptors, individually or in various combinations, which demonstrated that the receptors exhibit vital, but redundant, functions in fetal development and in vision, reproduction, and other functions required for maintenance of adult life. More recent advancements in sequencing and proteomic technologies reveal the complexity of retinoic acid receptor involvement in cellular function through regulation of gene expression and kinase activity. Future directions will require systems biology approaches to decipher how these integrated networks affect human stem cells, health, and disease.

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.

AU-rich sequence motifs (specifically sequences containing reiterations of AUUUA) are found in the 3' untranslated region of mammalian mRNAs encoding cytokines, adhesion molecules, and protooncogenes. Because these AU-rich elements (3'AURE) have been observed to reduce the stability and translational efficiency of transcripts that contain them, and because many of these transcripts accumulate in cells exposed to inflammatory stimuli, we reasoned that mRNAs with 3'AURE may be highly conserved and that the AURE is a marker of mRNAs that are inducible by environmental stressors. To test this hypothesis, we developed a polymerase chain reaction (PCR) strategy to isolate specifically mRNAs with 3'AURE. We first validated the effectiveness of this approach by selectively amplifying two mRNAs containing 3'AURE from interleukin 1 (IL-1)-induced human endothelial cells, then used the same primers in reverse transcriptase-PCR of sea urchin RNA, and used the radiolabeled reaction products to screen a cDNA library prepared from endotoxin-exposed sea urchin coelomocytes. We identified 124 positive clones and isolated a 1608-base-pair fragment that contains an AU-rich consensus sequence upstream from a poly(A) tail. This sea urchin transcript hybridizes with immobilized poly(A)(+)-selected RNA prepared from living coelomocytes maintained in vitro for 8.5-13 h but not with RNA prepared from freshly harvested coelomocytes. Our results provide support for the growing body of evidence that 3' AURE are both conserved and functional and indicate further that isolation and short-term in vitro culture of sea urchin coelomocytes is sufficient to induce the expression of transcripts containing 3'AURE.

Calcium protein placental homolog (Capl) is an S-100-related calcium-binding protein selectively expressed in cell lines that have been induced to grow or differentiate. In addition, the expression of Capl correlates with the induction of the metastatic phenotype in tumor cell lines and the transformation of normal cells by activated oncogenes or chemical carcinogens. Although not previously associated with the nervous system, in this study, Capl was purified from bovine neural retina by a combination of phenyl-Sepharose and organomercurial chromatography. The complete amino acid sequence of bovine Capl was established primarily by Edman degradation of peptides generated by cleavage of methionyl, lysyl, glutamyl, and aspartyl bonds. NH2-terminal methionyl and aspartyl peptides were analyzed by tandem mass spectrometry, which provided the sequence of the first 8 residues and identified the NH2-terminal blocking group as an acetyl moiety. The molecular mass of the intact protein determined by electrospray mass spectrometry (M(r) = 11,716.75 +/- 0.42) and the calculated molecular mass deduced from the amino acid composition (M(r) = 11,718) were in agreement, thus supporting the accuracy of the sequence assignment. Capl isolated from the retina was shown to be indistinguishable by mass and immunochemical properties from its counterpart in the bovine aorta, which previously was the only source of purified Capl. Northern analysis using cloned Capl cDNA revealed that Capl mRNA is present not only in the retina but the choroid as well. Further support for choroidal localization came from immunohistochemical experiments using specific anti-Capl antibodies. The physiological significance of Capl in ocular tissues and the aorta is discussed.

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.

Heart ventricles from chick embryos incubated in 60% O2 (hyperoxia) on the 16th through the 18th days of incubation were 21% heavier than those from control embryos maintained in 21% O2 (normoxia). Heart ventricles from embryos incubated in 15% O2 (hypoxia) were 8% lighter than controls. Changes in ventricular weight were accompanied by proportional changes in protein content (21% more in hyperoxic ventricles; 8% less in hypoxic ventricles). Ventricular tissue DNA content showed a significant increase in hyperoxia. Tissue protein/DNA ratios were significantly higher in hyperoxia and lower in hypoxia. These data suggest that increased O2 availability led to hypertrophy of chick embryo ventricular cells and an increase in the level of DNA synthesis. Cytochrome oxidase activity per mg DNA was 15–25% higher in hyperoxic ventricles than in hypoxic ventricles. This result is consistent with our previous findings that alterations in O2 availability affect the O2 consumption rate of the chick embryo in ovo, and it provides direct evidence that a phenomenon repeatedly observed in vitro is of importance in vivo. In contrast to the heart, O2 availability did not affect the wet weight, protein or DNA contents, or cytochrome oxidase activity of the chick embryo brain.

The hypothesis that oxygen availability limits growth of the normal chick embryo late in development predicts that an increase in oxygen availability would accelerate the rate of growth and, therefore, metabolism. We tested the prediction concerning metabolism by comparing the oxygen consumption (V O 2 ) and carbon dioxide production (V CO2 ) of 14–18 day embryos acutely exposed to either 60% or 100% O 2 with those of normoxic (21% O 2 ) controls. Two hours of hyperoxia produced increases in both V O 2 and V CO 2 ; however, repeated measurements over time in normoxia also demonstrated a significant increase in gas exchange, presumably due to normal growth of the embryos. After correcting for the increase in V O 2 due to growth, there was no effect of 60% O 2 on day 14. Thereafter the stimulatory effect of 60% O 2 increased gradually, reaching 6.1% on day 18. V CO 2 was 4 to 6% higher in embryos acutely exposed to 60% O 2 than in normoxic controls throughout the observation period, although the difference was significant only to day 18. The V O 2 of embryos acutely exposed to 100% O 2 was not significantly different from that observed in 60% O 2 , and was still significantly elevated 3 h after the eggs were returned to 21% O 2 . We conclude that acute hyperoxia late in incubation elicits an increased in embryonic V O 2 and V CO 2 , with little or no effect on the respiratory exchange ratio, and that the stimulation of gas exchange by 100% O 2 persists after the embryo is returned to normoxic conditions. These findings support the hypothesis that oxygen availability limits growth and metabolism of the normoxic chick embryo late in development.