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The Journal of Nutrition Vol. 128 No. 2 February 1998,
pp. 455S-458S
Department of Food Science and Human Nutrition, Michigan State University, East Lansing, MI 48824-1224
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ABSTRACT |
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Abstract
Introduction
References
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Vitamin A is an essential micronutrient throughout the life cycle. Its active form, retinoic acid via retinoid receptors, is involved in signal transduction pathways regulating development. Both the lack and excess of vitamin A during embryonic development result in congenital malformations. Approaches to examine the function of vitamin A in embryonic development have included treatment with excess retinoids and the use of retinoid receptor knock-out mice, which have provided important insights into the complexity of the retinoid signaling system. A recently explored model is the retinoid ligand knock-out, i.e., the vitamin A-deficient embryo. Early development can be successfully examined in the vitamin A-deficient avian embryo, in which bioactive retinoids can rescue the deficient genotype as well as phenotype. In this model it has been possible to unequivocally link the physiological function of vitamin A to development of heart, embryonal circulatory and central nervous systems and the regulation of heart asymmetry. Several developmental genes regulated by endogenous vitamin A during early embryogenesis have been identified. Retinoid receptors and their endogenous ligands, the vitamin A-active forms, are present in the early embryo. It is the developmentally regulated biogeneration of the vitamin A-active forms via distinct spatio-temporal metabolic pathways that is critically linked to the initiation of retinoid signal transduction during embryonic development.
KEY WORDS: vitamin A-deficient · embryogenesis · avian · heart · asymmetry
The essentiality of vitamin A for growth, vision, reproduction, embryonic and fetal development and tissue maintenance has been well established in numerous studies (Moore 1957 It was recognized already in the 1930s that maternal insufficiency of vitamin A during pregnancy results in fetal death and severe congenital malformations (Hale 1937 A recent approach to answering questions about the functions of vitamin A in development has been the use of transgenic mice with changes in retinoid receptor gene structure (Boylan et al. 1995 Another important approach to examine the molecular mechanisms of retinoid action in developmental regulation is the use of in vivo vitamin A-deficient animals, i.e., retinoid ligand knock-out models, in which gene expression can be studied by regulating the presence of the receptor ligands, the retinoids. Using a rodent model, it is possible to address the role of vitamin A in fetal development (Smith et al. 1998 Teratogenic effects of vitamin A deficiency and excess both involve heart morphogenesis, and retinoid receptor knock-out data provide a molecular basis for these abnormalities. Our laboratory has begun to investigate the function of vitamin A in early heart development using the vitamin A-deficient quail model. Early embryonic heart development in the avian is comparable to that of the human, and many analogies may be drawn from such studies. This is particularly relevant because congenital heart abnormalities in the Western industrialized world are as high as 12 per 1000 live births (Hoffman 1995 The rescue of the vitamin A-deficient embryo by the administration of bioactive retinoids, including retinoic acid, and the demonstration that the vitamin A-deficient phenotype can be induced in normal embryos by treating them with an anti-retinoic acid monoclonal antibody (Twal et al. 1995 In the last decade, significant advances have been made in identifying various genes involved in heart formation (Doevedans and VanBilsen 1996 Our studies with vitamin A-deficient quail embryos have revealed an intriguing fact: vitamin A is required for the normal specification of heart left-right asymmetry. This important developmental event seems to be critically dependent on the presence of vitamin A: in the vitamin A-deficient quail embryo the heart is on the wrong side, i.e., situs inversus in 75% of the cases (Twal et al. 1995 In the normal embryo, distinct developmental events are likely initiated by the presence of vitamin A-active molecules and further fine-tuned through the diversity of receptor combinations as well as by different retinoid ligands activating specific genes in a distinct spatio-temporal framework. Clearly, the function and metabolism of vitamin A are inseparable. Direct measurements of embryonic endogenous retinoids have been performed by HPLC in various species (reviewed in Costaridis et al. 1996 I thank the Department of Animal Science, Michigan State University and R. Balander and A. Napolitano of Poultry Research for providing for the use of the facilities on the Poultry Research Farm and for their continued advice and support of the project, and Hoffmann-LaRoche Inc., Nutley, NJ, for their generous support of this research.
INTRODUCTION
Abstract
Introduction
References
, Wolf 1984). However, the molecular mechanism(s) of action of this pleiotropic micronutrient are still under intense investigation. In the last two decades, much has been learned about vitamin A function from cell and molecular biology research and developmental studies. The concept of vitamin A as a powerful differentiating agent evolved from the early studies of vitamin A-deficient rats (Wolbach 1954) and was later substantiated by tissue culture studies (Fell and Mellanby 1953) and subsequent extensive research with cells, organ culture and animal models (Brockes 1989, Gudas et al. 1994). The regulation of somatic functions by vitamin A is now generally ascribed to retinoic acid, the biologically most active form of vitamin A, a recognized regulator of cell division and differentiation in tissues of ectodermal, endodermal and mesodermal origin (Gudas et al. 1994, Roberts and Sporn 1984, Wolf 1984). However, it was the discovery of the nuclear receptors for retinoic acid, the RARs, and the retinoid-X-receptors, the RXRs, that provided a breakthrough in the understanding of how vitamin A-active molecules can exert an effect at the gene level (Mangelsdorf et al. 1995, Pfahl and Chytil 1996). Retinoic acid and other retinoids
a collective term for vitamin A-related compounds of natural and synthetic originare capable of producing alterations in the expression of a diverse group of genes (Gudas et al. 1994). These findings have opened new avenues for the elucidation of the many and seemingly conflicting biological effects of vitamin A. Retinoic acid is now generally recognized as an important signaling molecule that as a ligand to its nuclear receptors, the RARs, alters gene expression at the level of transcription (Gudas et al. 1994, Mangelsdorf et al. 1995, Pfahl and Chytil 1996, Roberts and Sporn 1984). Another endogenous vitamin A-active molecule is 9-cis-retinoic acid, which can activate both the RARs and RXRs (Leblanc and Stunnenberg 1995). It is important to keep in mind that the function of vitamin A in the visual cycle is via a non-genomic mechanism involving retinal as a prosthetic group on visual proteins (Wald 1968).
, Mason 1935). In subsequent studies, Wilson and co-workers identified a spectrum of congenital abnormalities that result from lack of vitamin A during gestation (reviewed in Thompson 1969, Wilson et al. 1953). The major target tissues of vitamin A deficiency include the heart, the ocular tissues, and the circulatory, urogenital and respiratory systems. Including vitamin A in the diet of the female during specific times of pregnancy prevented the occurrence of these abnormalities, suggesting that vitamin A is required at various distinct stages of development and clearly establishing the important role of vitamin A in normal embryonic development. Embryonic malformations also result from the presence of excessive amounts of vitamin A during development, as was established using pharmacological levels. Cohlan (1954) was the first to describe congenital malformations in rats caused by the administration of excess vitamin A during pregnancy. Numerous studies and clinical observations of the teratogenic effects of excess retinoids have followed. Major target tissues include the heart, skull, skeleton, limbs, central nervous system, brain, eyes and craniofacial structures (Brockes 1989, Kochhar and Christian 1997, Kochhar et al. 1993, Moore 1957, Morriss and Steele 1972, Nau et al. 1994, Rosa 1993, Shenefelt 1972). Because of congenital abnormalities resulting from prenatal exposure to retinoids (Kochhar et al. 1993, Lammer et al. 1985, Rosa 1993), the use of retinoids is not allowed during pregnancy or when there is a possibility of pregnancy (Chan et al. 1996). The overlap of the teratological symptoms of vitamin A deficiency and excess indicates common targets and a critical role for vitamin A in the development of many organs. However, approaches using concentrations well above endogenous levels may not accurately reflect the physiological function(s) of vitamin A in the normal developmental program.
, Chambon 1993, Giguere et al. 1996, Kastner et al. 1994, Sucov et al. 1994). Many of the abnormalities in these mutant mice resemble those observed in the fetuses from the vitamin A-deficient animals reported earlier. The molecular and genetic dissection of the retinoid signaling pathway has provided valuable insights into the pleiotropic effects of vitamin A and demonstrates the critical role of retinoid receptors in vertebrate ontogenesis. However, the interpretation of the data has been hampered by receptor redundancy (Luo et al. 1995). Thus the receptor knock-outs alone can not provide definitive answers to retinoid function. These studies, however, underscore the critical importance of the retinoid ligands: evolution has provided a redundancy of receptors to serve as back-up systems for the essential functions of vitamin A.
, Thompson 1969). An ideal model to study the role of vitamin A in very early development is the vitamin A-deficient avian embryo, which is completely dependent on vitamin A for its early cardiovascular development and dies at d 3 of embryonic life (Dersch and Zile 1993, Thompson 1969). Heine et al. (1985) determined that vitamin A is required for establishing the cardiovascular circulatory system. Our laboratory has further developed this avian model to study the function of vitamin A in early embryonic development. Retinoic acid provided as the only source of vitamin A to the adult quail is not transferred to the egg (Chen et al. 1996); thus a completely vitamin A-deficient embryo can be obtained (Dong and Zile 1995). The vitamin A-deficient quail embryo is grossly abnormal, not only in respect to its cardiovascular system but also in its early development of the head, central nervous system, haematopoiesis and trunk (Dersch and Zile 1993, Maden et al. 1996 and 1998, Twal et al. 1995, Zile et al. 1997a). It is possible to "rescue" the vitamin A-deficient embryos, i.e., to prevent the abnormal development, by administering bioactive retinoids during very early stages of development (Dersch and Zile 1993, Heine et al. 1985, Sporn et al. 1985, Thompson 1969, Zile et al. 1997a).
), and pediatric cardiovascular abnormalities account for 8% of all deaths during the first year of life. At this time there is no understanding of the molecular basis of these malformations. However, more and more clinical data confirm that it is important to look at the nutritional and dietary contributions to birth defects. Poor diet or harmful dietary components may be ingested during pregnancy, a time when vitamin A nutrition is very critical because maternal tissues are continuously being depleted of vitamin A so as to ensure an adequate supply of it to the embryo and fetus (Wallingford and Underwood 1986). Although nutritionists are well aware of the marginal vitamin A status in the developing countries (Underwood 1994), little is known of populations at risk in the industrialized countries. The study by Duitsman et al. (1995) is significant because it identifies a population of pregnant U.S. women at high risk of vitamin A inadequacy. Because the majority of embryonic deaths occur very early in pregnancy, information is not available as to their possible causes. The critical vitamin A-dependent developmental events are initiated during gastrulation and neurulation and coincide with the first 2-3 wk of human pregnancy. They may be severely compromised if maternal vitamin A intake is marginal or if there is an interference with vitamin A function. It is clear from our studies with the avian embryo model that there is an absolute requirement for vitamin A during the very beginnings of life, when the absence of vitamin A results in gross abnormalities and early embryonic death.
) confirm the involvement of retinoic acid or an immediate metabolite of it in the regulation of early embryonic development and link these developmental events to retinoid receptors. Retinoid receptors are known to be expressed in the early embryos of all species examined (reviewed by Hofmann and Eichele 1994). Smith and Eichele (1991) and Smith (1994) examined the expression of RAR
in the normal avian embryo; only limited data exist on the expression of these genes in the vitamin A-deficient embryo. Kostetskii et al. (1996) observed a significant decrease in the expression of RAR2 in the heart-forming areas of the vitamin A-deficient quail embryo, whereas the expression of RAR
and RAR
as well as RXR and RXR was not affected by vitamin A deficiency (Zile et al. 1997b). The presence of RAR in the presumptive cardiogenic mesoderm of chick was also demonstrated earlier by Smith (1994), suggesting that retinoids may direct mesoderm into heart lineage. Our observations of a spatio-temporal coexpression of RXR and RXR with RAR2 in the cardiogenic regions of the early quail embryo (unpublished data) suggest that these retinoid receptors are involved in the retinoid-regulated heart-forming events.
, Kern et al. 1995). The absence of master regulators in the heart implies the importance of interactions among many factors. Among the few transcription factors expressed predominantly in the heart are the GATA-4, 5 and 6, a subfamily of genes recently identified in the chick (Doevedans and VanBilsen 1996, Laverriere et al. 1994). We have observed that the expression of GATA-4 is significantly decreased in the heart-forming regions of vitamin A-deficient embryos and that this expression can be induced by the administration of vitamin A to the embryo prior to heart formation (unpublished observation). Retinoid-regulated heart-forming events may also involve Msx-1, a gene of the homeobox class of transcription factors (Kern et al. 1995). Msx-1 expression was significantly altered at various sites, including the heart-forming region of vitamin A-deficient quail embryos (Chen et al. 1995).
, Zile et al. 1997a). Our work has shown that as late as neurulation stage 8 (but not later) the anticipated vitamin A-deficient phenotype, including cardiac situs inversus, can be "rescued" by administration of vitamin A-active compounds (Dersch and Zile 1993, Zile et al. 1997a). This is a challenging observation because it can not be explained by the recently proposed molecular pathway determining cardiac left-right (L-R) asymmetry (Levin et al. 1995), which encompasses developmental stages prior to our observed vitamin A-sensitive window for cardiac L-R asymmetry regulation. Cardiac abnormalities also result when embryos are treated with excess retinoids (Hofmann and Eichele 1994, Osmond et al. 1991, Shenefelt 1972). Duplicate hearts, cardia bifida, abnormal looping and situs inversus can be produced by implanting excess retinoic acid-containing microbeads in specific precardiac regions of chick embryos (Dickman and Smith 1996, Osmond et al. 1991). Although these experiments do not provide complete answers to the normal physiological mechanism of action of vitamin A in cardiogenesis, they point to embryonal sites, such as the heart-forming areas, that are very sensitive to exogenous retinoic acid and that are also likely to be sensitive to the lack of this signaling molecule, as shown in the vitamin A-deficient quail. Recently, extracellular matrix (ECM) proteins with cardiac specific L-R localization have been identified by their respective antibodies LAMP 1 (Smith et al. 1997) and F-22 (which identifies a large ECM protein flectin; Tsuda et al. 1996). Flectin distribution in the heart-forming regions is predominantly on the left side and is affected by vitamin A status. A fibrillin-like molecule that is recognized by the monoclonal antibody JB3 is localized asymmetrically in early heart-forming regions of chick embryos (Smith et al. 1997, Wunsch et al. 1994); its location is reversed after treatment with exogenous retinoic acid (Smith et al. 1997). The genes of the above ECM molecules may participate in regulating heart laterality.
, Hofmann and Eichele 1994, Horton and Maden 1995). HPLC analysis of endogenous vitamin A metabolites in the neurulation stage quail embryo revealed the presence of the bioactive RAR ligands all-trans-retinoic acid and didehydroretinoic acid as well as their precursors retinal and retinols and the storage forms, retinyl esters (Dong and Zile 1995). Clearly, the early embryo has the enzymatic capabilities to fully process vitamin A. It is important to keep in mind that the developing embryo is very sensitive to a lack as well as an excess of retinoids. From studies with young and adult animals, it is well known that circulatory vitamin A level is strictly regulated within a narrow range, and that relatively small amounts of retinoids exist in their free forms (Blaner and Olson 1994, Wolf 1984), suggesting a stringent homeostatic regulation of the availability of bioactive retinoids. In the embryo, such regulation must be under developmental control. The biogeneration of vitamin A active form(s) in the embryo is likely the initial and critical developmental event in the initiation of the retinoid-regulated signaling pathways, inseparably linking the function of vitamin A to its metabolism. The regulation of biosynthesis of the vitamin A active form, retinoic acid, during embryonic development in the mouse is being examined and reviewed by Duester (1998) and Dräger et al. (1998). This symposium highlights many of the approaches taken by scientists to understand how developing embryos biogenerate and use vitamin A active forms and what goes wrong when there is a deficiency in this important vitamin.
ACKNOWLEDGMENTS
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FOOTNOTES |
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LITERATURE CITED |
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Abstract
Introduction
References
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developmental function: convergence of RXR and RAR signaling pathways in heart and eye morphogenesis.
Cell
1994;
78:987-1003 [Medline] [Medline] develop normally and are susceptible to the teratogenic effects of retinoic acid.
Mech. Dev.
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53:61-71 [Medline] [Medline]2 is an early marker for alimentary tract and central nervous system positional specification in the chicken.
Dev. Dyn.
1994;
200:14-15 [Medline] [Medline]-transcripts in the chick embryo.
Development
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111:245-252 [Medline] [Abstract] mutant mice establish a genetic basis for vitamin A signaling in heart morphogenesis.
Genes Dev.
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