I. INTRODUCTION

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Vitamin A and its derivatives (the retinoids) are essential for both normal embryonic development (87) and maintenance of differentiation in the adult organism (35, 36, 78), accounting for the intense interest in these compounds in biology and medicine. Embryo segmentation and growth fail, vascularization stops, and the embryo is eventually resorbed in the absence of sufficient vitamin A (151, 279, 297). Excess vitamin A, on the other hand, results in terata during embryogenesis and is membranolytic and hepatotoxic in the adult organism (204). In the adult, epithelial differentiation requires vitamin A. Its deficiency causes squamous metaplasia, a preneoplastic lesion, which eventually also alters functional epithelial characteristics and leads to infection and death (36, 247, 306). These considerations make it obvious that maintenance of retinoid homeostasis is tantamount to maintenance of normal physiology of the intact organism.

The use of the retinoid ligand knockout models to study embryonic development has unequivocally linked the physiological function of vitamin A to development of the heart, the embryonal circulation, the central nervous system (CNS), and the normal left-to-right cardiac symmetry (321). Recent work has also emphasized that the developmentally regulated generation of bioactive retinoids is fundamental to the control of embryonic development (47, 321).

Remarkable progress has occurred in the past 10 years in our understanding of the mode of action of vitamin A and its derivatives, the retinoids (37). The discovery of the nuclear receptors for retinoic acid and other retinoids has provided a conceptual basis to explain how these compounds preside over a large network of gene activation processes (35).

It is the purpose of this review to present a balanced synopsis of the actions of retinoids in embryonal development and to provide suggestions for future studies.

	II. RETINOID STRUCTURE

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Figure 1 shows the chemical structure of the salient, physiologically important retinoids so far identified. The parent compound retinol and its oxidation product, retinoic acid (RA), are shown in the stretched-out all-trans-configuration at the top of the figure. Derivatives of retinol include 4-hydroxy-retinol, 4-oxo-retinol, and 14-hydroxy-4,14-retro-retinol. Of the RA derivatives, 4-hydroxy-RA, 4-oxo-RA, 3,4-didehydro-RA (ddRA), and the rexinoid receptor (RXR) ligand 9-cis-RA are shown. All-trans RA is the natural ligand for the retinoic acid receptors (RAR) (71, 221), and 9-cis-RA for the RXR (137), although the latter compound binds to both receptor families. The specificity of interactions would also suggest the possibility that various retinoids, both natural and synthetic, may specifically be useful as drugs to combat diverse diseases.

View larger version (30K): [in this window] [in a new window]   Fig. 1. Structures of the most common natural retinoids.

	III. RETINOID REQUIREMENT DURING EMBRYOGENESIS

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Retinol (also referred to as ROL and/or vitamin A) is the only retinoid known to be capable of sustaining all vitamin A functions, including development, growth, vision, and reproduction (Fig. 2). On the other hand, RA maintains differentiation and growth of the adult organism; it, however, is insufficient to support gestation of the embryo. Because RA cannot fulfill all vitamin A functions, it is obvious that several retinoids, in addition to RA and which are not RA metabolites, act in concert to maintain the health and well being of the entire organism. Also possible are limitations of cellular metabolism and transport, where cells require RA but must transport retinol across a tissue barrier. For a more detailed discussion of the different roles of vitamin A, the reader is referred to the following reviews (11, 263, 307).

View larger version (83K): [in this window] [in a new window]   Fig. 2. Highlights of the various functions of vitamin A in males and females.

As early as the 1930s it was realized that maternal insufficiency of vitamin A results in death of the fetus as well as congenital malformations (79, 172). Later, Wilson and co-workers (279, 305) defined congenital abnormalities resulting from vitamin A deficiency during gestation. Teratogenic targets of vitamin A deficiency were the heart, ocular tissues, and respiratory, urogenital and circulatory systems (321).These abnormalities were prevented by inclusion of vitamin A in the diet.

Excess dietary vitamin A, on the other hand, has been shown to also cause teratogenesis (26). Several other studies followed this original observation and are discussed in section VIII. Suffice it to say here that major excess vitamin A targets include the heart, the skull, skeleton, limbs, brain, eyes, CNS, as well as craniofacial structures (15, 110, 111, 189, 191, 203, 234, 252, 322).

Similarity of teratogenic responses between vitamin A deficiency and excess supports the concept that the same molecules are involved. It also suggests a fundamental role for this nutrient in embryonal development.

The work of Wellick and De Luca (297) has demonstrated that retinol is essential and RA alone appears insufficient to allow gestation in the rat to reach completion. In fact, vitamin A-deficient pregnant rats resorb their fetuses at about day 15 of gestation , even if given retinoic acid daily. Retinol, on the other hand, prevents this resorption when administered no later than day 10 of gestation. In fact, the administration of as little as 2 µg on day 10 is sufficient to allow continuation of gestation through parturition (298). Claggett-Dame recently proposed that extremely high RA doses may, in fact, rescue the dpc 14.5 gestational barrier identified by Wellick and De Luca (298). Whether this reflects rescue by traces of alternative retinoids in not known at this time. Countering this is Niederreither's RALDH2 knockout, which is incompletely rescued by exogenous RA (206, 207).

The recommended dietary allowance (RDA) for vitamin A for nonpregnant and pregnant women is 800 µg RA, which is equivalent to ~2,700 IU (210). No increment of vitamin A is recommended during pregnancy (210, 214). For a discussion of the data which justify this recommended intake, the reader is referred to the 10th edition of the RDA (210).

Different retinoid requirements between the avian and mammalian systems have been highlighted and are summarized as follows: 1) ddRA and its precursor 3,4-didehydroretinol (ddROH) were undetectable in mouse limb buds, although prominent in chick limbs; 2) a relatively high concentration of retinyl esters (1.5 µM) is evident in chick limb buds but not in mouse; and 3) there is a higher concentration of cellular retinoic acid binding proteins (CRABP), especially CRABPII, than their ligands RA and ddRA in both species. Retinol and ddROH, on the other hand, were present in much higher concentration than cellular retinol binding proteins (CRBP) (245). The concentration of "free" RA was found to be near the range for the dissociation constant (K_d) value of the murine RAR.

Principal approaches to study the essential function of vitamin A during embryonal development are exposure to excess retinoid, retinoid receptor knockout studies, and finally retinoid-ligand knockout models. This last approach is discussed in section IIIB.

The studies of Zile (321) have demonstrated the usefulness of the vitamin A-deficient (retinoid ligand knockout) avian embryo. These embryos display a strict dependence on vitamin A for their development of early vasculature. These avian embryos die at 3 days of embryonic life in the absence of vitamin A (43, 279). After the initial discovery by Heine et al. (83) that vitamin A is necessary for the establishment of the early vasculature, Zile (321) extended these studies to early development. Dong and Zile (53) and Chen et al. (25) had established that RA given to the hen is not transported to the egg. In this way a completely vitamin A-deficient embryo can be obtained from hens that are RA sufficient (53). This model was used by Zile and collaborators to define primary target tissues of vitamin A action during embryogenesis.

The vitamin A-deficient quail embryo presents with gross abnormalities in the cardiovascular system, head, CNS, hematopoietic organs, and trunk (43, 151, 152, 283, 321, 322). Bioactive retinoids can "rescue" the vitamin A-deficient embryo by preventing abnormal development when available at early development (43, 83, 115, 117, 279, 321). Because both retinoic excess as well as deficiency had similar teratogenic effects on the heart, Zile's group has launched an in depth investigation of the function of vitamin A in early heart development, using the vitamin A-deficient quail model (115-117). These studies are of particular interest because of the obvious relevance to human cardiovascular malformations, which may well be diet-related during pregnancy. Availability of vitamin A may be a relevant contributory element to the incidence of heart malformation in developing countries, where vitamin A deficiency is a significant problem (261). Very little is understood of its etiology in the industrialized world, hence, the importance of better definition of etiological factors, especially dietary ones during pregnancy, since diet is likely to contribute the majority of xenobiotic as well as homobiotic factors to the physiology and pathology of the body. Vitamin A is also required for normal specification of heart left-right asymmetry. In a large percentage of vitamin A-deficient quail embryos, the heart appears on the wrong side (randomization) (281, 283, 284, 323). Retinoids, although not directly involved in assigning cardiac asymmetry genes to their asymmetry-specific sites, are absolutely essential for normal heart sidedness to occur (M. H. Zile, personal communication). Importantly, administration of vitamin A to deficient embryos as late as stage 8 (neurulation) prevents the anticipated vitamin A-deficient phenotype, including situs inversus (43, 115, 117, 323). They propose that the critical retinoid-requiring developmental window is at the four/five-somite stage during neurulation, when retinoid presence is absolutely essential for normal embryonic development to proceed. Excess retinoid resulting from implantation of retinoid impregnated pellets also causes cardiac abnormalities, including duplicate heart, cardia bifida, abnormal looping, and situs inversus (46, 215). Genes coding for extracellular matrix proteins appear important during heart embryogenesis and are regulated by retinoid status (267). Kostetskii and collaborators (116, 117) have found that RAR2 expression in the retinoid ligand knockout quail model is reduced, as is RAR. They also reported that several retinoids, including all-trans-, 9-cis-, 4-oxo-, and didehydro-RA could rescue embryonic development, when provided to the developing embryo at up to the five-somite stage of development, but not later. These retinoids induced RAR expression; 4-oxo-RA is least active.

Metabolic routes of retinoid activation and disposal are highly relevant to the occurrence of orderly developmental events and are covered separately in this review.

Rat embryos made deficient in retinoids during embryogenesis (gestational days 11.5-13.5) show specific cardiac, limb, ocular, and nervous system deficits (258). Many of these abnormalities also occur in retinoid receptor null mutants as discussed in section VI. Retinoid-ligand knockout models reveal additional defects, however, clearly supporting redundancy of the retinoid receptors (258).

Stratford et al. (267) have recently identified target genes in the development of the early limb bud of the quail, in the retinoid ligand knockout model. The retinoid-deficient quail embryos were found to develop to about stage 20/21. In a study of genes involved in anteroposterior axis, these authors found that Hoxb-8 was upregulated and its border was shifted anteriorly. In sharp contrast, shh and mesodermal bmp-2 were downregulated. These authors also studied the apical ectodermal genes and found that fgf-8 and ectodermal bmp-2 were not affected (267). A strong effect of retinoid deficiency was observed for genes involved in dorsoventral polarity. Wnt-7a was expressed in ventral ectoderm, although normally it is expressed exclusively in dorsal ectoderm. On the other hand, the corresponding dorsal mesodermal gene Lmx-1 was found to extend its expression into the ventral mesoderm. En-1 expression was absent from its normal expression site, the ventral ectoderm (267). This study exemplifies the usefulness of this model to study the role of retinoids in the regulation of gene expression during development.

Kostetskii et al. (115) have investigated the expression of cardiomyocyte differentiation genes. They have reported that vitamin A deficiency in the quail embryo fails to alter these genes including atrial-specific myosin heavy chain, ventricular-specific myosin, and sarcomeric myosin as well as the putative cardiomyocyte specification gene Nkx-2.5. However, the expression of the transcription factor GATA-4 was greatly reduced in the heart-forming region of stage 7-10 embryo in vitamin A deficiency. This downregulation occurs in the areas of the heart which eventually progresses to develop abnormally in the absence of vitamin A, the lateral mesoderm posterior to the heart. These authors also report that administration of retinol to the vitamin A-deficient embryos is able to restore GATA-4 expression and completely rescues the vitamin A-deficient phenotype. The authors conclude that their results indicate GATA-4 is a component of the retinoid-mediated cardiogenic pathway. They also conclude that this pathway appears unlinked to the differentiation of cardiomyocytes, and it is involved in the morphogenesis of the posterior heart tube as well as the development of the cardiac inflow tract, which does not form in the vitamin A-deficient embryo.

	IV. METABOLISM

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RA regulates development by activating gene transcription in many different locations within the embryo. A particular cell will only respond to RA if 1) it expresses RA receptors and 2) the RA concentration lies within a range that is appropriate for its response. Because different genes are known to be activated by different RA levels (13), the effectiveness of RA as a developmental regulator involves the precise control of its distribution and concentration. Several mouse strains transgenic for a RA-activated reporter gene show that RA is not homogeneously distributed throughout the vertebrate embryo, but is instead localized to specific territories within a restricted number of developing organs in the embryo (8, 236). Studies on RA metabolism in the embryo indicate that the RA-synthesizing and catabolic enzymes are localized to subregions of developing tissues (175, 194, 206, 319), which resemble the activation patterns in the RA-reporter mice (8, 236). The pathways of synthesis and catabolism thus establish the local levels of RA and determine where and when RA regulation occurs. The most detailed description of this localization is available for the embryonic retina (54) and spinal cord (175). In addition, several other regions exhibit localized RA synthesis, both in the CNS, such as the substantia nigra and corpus striatum (176), and in mesoderm-derived tissues, such as the meninges (206), heart (194), and somites (206). Several regions in the developing CNS, e.g., the limb motor neurons in the spinal cord (319), express very high levels of RA-synthesizing enzymes, resulting in remarkably high RA concentrations locally, up to micromolar levels. The RA-rich regions can be located next to areas of high RA catabolism that contain little detectable RA, as is the case for the embryonic retina described in more detail below (181). The analysis of the distribution of RA synthetic and catabolic enzymes shows that several developing tissues contain compartments of high and low RA levels, a morphogenetic RA pattern in addition to, and different from, the earlier model of patterning through RA gradients (280).

The first step in RA synthesis is the reversible oxidation of retinol to retinaldehyde (22) (see Fig. 3). The full complement of enzymes that catalyze this reaction in the embryo remains to be determined. It is likely, though, that most of the retinaldehyde in the embryo is generated by alcohol dehydrogenases. A point of contention is, however, whether the medium-chain cytoplasmic alcohol dehydrogenases or the short-chain membrane-bound alcohol dehydrogenases are more important. Several members of the medium-chain, cytoplasmic class are competitively inhibited by ethanol, whereas the short-chain membrane-bound class is resistant to ethanol inhibition. The answer is important in reference to the hypothesis that the abnormalities evident in fetal alcohol syndrome result from the inhibition of RA synthesis by ethanol (58, 227, 315). This hypothesis has been proposed, because of the similarities between the embryonic abnormalities evident in RA teratogenicity and in fetal alcohol syndrome.

View larger version (11K): [in this window] [in a new window]   Fig. 3. The metabolic flow from retinol to retinoic acid and its oxidation products is shown. Note that the oxidation of retinal to retinoic acid and further oxidation reactions are irreversible.

This family of enzymes consists of at least four classes of zinc-dependent cytosolic enzymes: classes I, II, III, and IV (Adh1, -2, -3, and -4). Both Adh1 and Adh4 dehydrogenases can oxidize retinol to retinaldehyde, but only the Adh1 enzymes are inhibited by ethanol. In the adult, ethanol has been shown to inhibit RA synthesis in several tissues, including testes (289), liver (186), and cornea (94). Moreover, the alcohol dehydrogenase specific inhibitor 4-methylpyrazole has been shown to inhibit retinaldehyde synthesis in the testes (289). It is clear, however, that several retinol dehydrogenases are neither competitively inhibited by ethanol nor inhibited by 4-methylpyrazole. The alcohol dehydrogenase negative deermouse lacks all ethanol dehydrogenase activity but can oxidize retinol normally (226). Because this strain of deermouse is apparently normal, the ethanol-sensitive form of retinol dehydrogenase does not seem to be essential for normal development. The house mouse expresses only a single gene of the Adh1 group, and although its human homolog contains a RA response element, this is not the case in the mouse (292). In the mouse embryo, Adh1 is distributed in only a few of the regions known to synthesize RA, such as the developing kidneys (236, 292), and it is entirely absent from the CNS (292). It seems thus unlikely that Adh1, the only enzyme in the mouse competitively inhibited by ethanol, is important for RA synthesis during most of embryogenesis. Another candidate for embryonic retinol dehydrogenase is Adh4, since it oxidizes retinol to retinaldehyde and is expressed in several regions of embryonic RA synthesis (4). In particular, its expression commences at embryonic day 7.5, the time when RA synthesis begins (236). It is present in the early CNS and also in the tissue around the developing eye, both regions of high RA synthesis, as well as the otic vesicles and migrating neural crest (81). Adh4 is, however, absent from the embryonic retina at all times and from the entire embryo after embryonic day 10.5 (4); hence, retinaldehyde for embryonic development from this stage onward must be synthesized by another enzyme. In addition, null mutations for Adh4 , as well as for Adh1, develop normally (34), indicating that, even at early developmental stages, redundancy in the retinol dehydrogenases exists.

At least five microsomal retinol dehydrogenases of the short-chain dehydrogenase/reductase are known to be expressed in the adult (270). These enzymes are typically resistant to inhibition by 4-methylpyrazole and ethanol. The type II microsomal dehydrogenase was reported to be associated with the cytochrome P-450/CYP2D1 oxidase which, combined with NADPH-P-450 reductase, promotes retinaldehyde oxidase activity (92). It is possible, then, that the short-chain dehydrogenase/reductases may be particularly important for retinaldehyde reduction to retinol, competing with the oxidation of retinaldehyde to bioactive RA. The embryonic distribution of the five dehydrogenase/reductases is largely unknown. Two recently identified short-chain dehydrogenase/reductases, however, have interesting properties; they are specific for cis-retinol isomers and do not catalyze all-trans-retinol oxidation (185, 233); they are named 9-cis-retinol dehydrogenases. One of the two was found to be broadly expressed in many regions of the embryo, including the CNS, eye, ear, and somites (233). The other one is identical to the 11-cis-retinol dehydrogenase required for regeneration of the visual chromophore in the retinal pigment epithelium (56, 256). In addition to the eye, it is expressed in several other tissues in the adult organism, including the testes, but its localization in the embryo is not yet known. The identification of a 9-cis-retinol dehydrogenases implies that 9-cis-RA, the only RA isomer that activates the RXR receptors, is generated from 9-cis-retinoid precursors, rather than by a RA isomerase from all-trans-RA. Such a route for 9-cis-RA synthesis is attractive, because a pool of 9-cis-retinol is known to exist, at least in the adult (124), and because a RA isomerase has remained elusive (285, 286). If RA isomerization does occur during normal development, then it may not be enzymatically catalyzed (285, 286), although tissues are known to differ in their ability to isomerize all-trans-RA to 9-cis-RA (131). It is also relevant that, so far, Xenopus is the only species in which 9-cis-RA has been identified in the embryo (118), although this may reflect lower levels of the isomer in embryos of other species.

The 9-cis/11-cis-retinol dehydrogenase is similar to the other short-chain microsomal enzyme in that it is insensitive to ethanol inhibition (185). An intriguing characteristic of type I, and perhaps other short-chain retinol dehydrogenases, is that its activity is stimulated by ethanol. This is consistent with the features of the mouse fetal alcohol syndrome (275, 295), which are more similar to the effects of a RA excess (252), than to the effects of vitamin A deficiency (305). We have evidence that some of the teratogenic actions of ethanol in the brain are due to the abnormal induction of RA synthesis (P. J. McCaffery and D. Ullman, unpublished observations).

The embryonic enzymes that catalyze the irreversible oxidation of retinaldehyde to RA are predominantly of the cytosolic class I aldehyde dehydrogenase family (22, 177). Although retinaldehyde oxidases exist (163, 232) and a proportion of rat embryonic RA synthesis is believed to occur via oxidases (22), our work (175, 194, 206) demonstrates that the retinaldehyde dehydrogenases are almost totally responsible for the RA distribution observable in the mouse embryo.

In our search for retinaldehyde dehydrogenases we initially employed a zymographic technique (178). For this assay, native tissue extracts are separated by isoelectric focusing (IEF), and retinaldehyde dehydrogenases are identified with RA-reporter cells by their capacity to oxidize retinaldehyde to RA in a NAD-dependent reaction. This bioassay is highly sensitive, allowing the use of very low substrate concentrations and detection of synthesized RA levels similar to those present in vivo. This feature enabled the detection of two previously unknown retinaldehyde dehydrogenases in the embryo, which we initially termed V1 and V2. The V2 gene is now cloned and has been renamed RALDH2 (319), while V1 remains to be characterized at the molecular level. A third enzyme present in the eye had already been described as the predominant enzyme for RA synthesis in the adult mouse liver (133); this third enzyme is referred to as AHD2 in the mouse, following its gene locus name.

The two enzymes AHD2 and V1 create the very high and spatiotemporally regulated RA levels in the embryonic retina. Apart from the embryonic retina, they are only expressed in a few restricted locations within the embryo, i.e., within the CNS. AHD2 is found only in a subpopulation of dopaminergic cells in the substantia nigra and adjoining ventral tegmentum, and within the head, V1 is only expressed in the maxillar regions of the developing face and the lateral ganglionic eminence of the brain (unpublished observations). In the retina (see Fig. 4), AHD2 is restricted to a dorsal (D) region, whereas V1 is expressed in the ventral (V) portion (179). AHD2 is relatively inefficient in synthesizing RA from the low concentrations of retinaldehyde present in the retina. In contrast, V1 shows a much greater efficiency at these retinaldehyde concentrations, and the net result is greater RA synthesis in the ventral than dorsal retina (179). It would be expected that the boundary region between the two enzymes contains an intermediate concentration of RA and that a ventral-to-dorsal gradient extends across the entire retina. Instead, between the two RA-synthesizing enzymes lies a horizontal stripe of the RA catabolic enzyme CYP26 (181). This arrangement creates three regions along the dorsoventral axis of the embryonic retina, a dorsal zone of intermediate RA concentration, an intervening region of very low RA and a ventral zone of high RA concentration (Fig. 4). Gradients of RA concentration may exist within each region. This is the first description of a mechanism by which zones of RA concentration can be created. Inhibition of RA synthesis by citral in zebrafish blocks the development of the ventral half of the retina in similar fashion to the effects of null mutations of the RA receptors (98, 140, 167). Thus the ventral retina is a zone distinguished by its expression of the V1 RALDH, by its high RA synthesis, and by its dependence on RA for development. The dorsal, medial, and ventral territories of differing RA levels are likely to regulate transcription of some of the genes with differential expression patterns along the dorsoventral axis of the retina. Such genes include the tyrosine receptor kinase EphB2 and its ligand ephrin B2, which are believed to influence the target selectivity of outgrowing retinal axons (164), and the green and blue cone photoreceptors in the mouse (276). The ligand ephrin B2 and the green cones are restricted to the dorsal retina, and the receptor EphB2 and the blue cones are located predominantly in the ventral retina. It is likely that other regions of the embryo contain a similar zonal patterning of RA, created by the juxtapositioning of RA synthetic and catabolic enzymes.

View larger version (64K): [in this window] [in a new window]   Fig. 4. The distribution of retinoic acid (RA) in the embryonic mouse retina. The AHD-2 retinaldehyde dehydrogenase is specific to the dorsal (top) region and is distributed as a gradient from dorsal tip to the boundary of the catabolic enzyme CYP26, creating a gradient of RA in this orientation. The boundary stripe of CYP26 degrades RA creating a zone of very low RA concentration. Below CYP26 the "V1" retinaldehyde dehydrogenase is expressed, possibly in a gradient from ventral (bottom) tip to the CYP26 boundary creating a second RA gradient in this orientation. The overall amount of RA is greater in the ventral half compared with the dorsal half.

Another intriguing property of the class I aldehyde dehydrogenases, in addition to their enzymatic function, is their affinity for several ligands of the nuclear receptor family. The promoter of the human ALDH1 gene contains a putative response elements for androgen (313), and the enzyme has been demonstrated to bind androgen (218). The corresponding cytosolic aldehyde dehydrogenase in Xenopus has been identified as the major thyroid-hormone binding protein in this species (312). We found that AHD2 in the embryonic dorsal retina and substantia nigra is present in amounts far exceeding the concentrations of the other retinaldehyde dehydrogenases (180). This suggests this enzyme may have a dual function, both synthesizing RA and also modulating the local concentration of other nuclear receptor ligands that can influence RA signaling in the embryonic retina.

The retinaldehyde dehydrogenase RALDH2 exhibits the greatest substrate specificity of the three dehydrogenases, and its distribution provides the most accurate guide to the localization of all-trans-RA in the embryo. RALDH2 is essential for normal development, and its knockout results in a complete failure of embryo survival and early morphogenesis (207). The trunk of the embryo is severely shortened, and the neural tube remains open, whereas the anterior region is less affected. Unfortunately, the embryos die before the effect on more differentiated structures can be observed. Studies on these structures await the creation of conditional knockouts.

A guide to the pattern of all-trans-RA signaling in the embryo is provided by the RA-reporter mice, a strain transgenic for -galactosidase controlled by a sensitive RA response element (RARE-lacZ) (27, 236). The expression of -galactosidase in a particular cell is the result of the balance of RA synthesis and catabolism in that region, as well as the capacity of the cell to respond to RA depending on its expression of RA receptors, coactivators, and corepressors. Given the number of factors on which -galactosidase expression depends, it is surprising that the distribution of RALDH2 (206) matches quite well with the distribution of -galactosidase in the RA reporter mice (236), including expression in the spinal cord, motoneurons, and developing eye. When examined in greater detail, there was a very good correlation, for instance, in the embryonic heart (194). This suggests that a significant proportion of the patterning of RA signaling in the embryo may rely on localized RA synthesis. Regions do exist, however, in which -galactosidase distribution does not correlate with RALDH2 expression, and this includes the RARE-lacZ induction in the telencephalic vesicles and nasolachrimal groove. This is likely to be the result of RA synthesis by other RALDH. Analysis of RA distribution by HPLC also indicates a pattern similar to that of RALDH2 (90). With the exceptions of the restricted regions of V1 and AHD2 expression, which include the retina, face, and the striatum (176), RALDH2 appears to be responsible for retinaldehyde oxidation in most other regions of early embryonic all-trans-RA synthesis. Within the embryonic CNS, the limb motor neurons in the spinal cord (175, 260, 319) represent major sites of RALDH2 expression, whereas most of the remaining CNS shows lower RA synthesis. It is of note, however, that the meninges surrounding the brain contain extremely high levels of RALDH2, and this potent source of RA may be important in brain development and plasticity (unpublished observations). For the developing cerebellar region, the meninges are likely to represent a major source of RA required for the differentiation of granule cells in the adjacent external granular layer. RALDH2 does not discriminate between all-trans- and 9-cis-retinaldehyde (M. Warren, personal communication); hence, this enzyme does not appear to influence the all-trans- and 9-cis-RA ratios.

D.  Synthesis of Other Bioactive Retinoids in the Embryo

1.  Didehydro-RA

Although RA is the predominant active retinoid in the embryonic mouse, this is not the case in several other vertebrate classes. In the chick, didehydro-RA was found to be the principal active retinoid in the developing limb bud (278), as well as in the developing spinal cord and the somites (156). Moreover, the likely precursor didehydro-retinol has been detected in the embryonic chick. The distribution of didehydro-RA in the chick embryo is similar to the distribution of all-trans-RA in the mouse. Furthermore, the patterns of RALDH2 expression in both mouse (206) and chick (10) are similar. It is thus likely that RALDH2 will oxidize didehydro-retinaldehyde to didehydro-RA, although this has still to be demonstrated directly. Didehydro-RA is also present in both Xenopus and zebrafish (29, 30) but has yet to be detected in mouse (90).

In the embryonic Xenopus (223), all-trans-4-oxo-RA influences the development of the anteroposterior body axis in a fashion that is unresponsive to all-trans-RA. Both retinoids act through RA receptors, and it is assumed that they activate differing configurations of receptors. Similarly, the 9-cis-isomer of 4-oxo-RA differs from 9-cis-RA in receptor selectivity, since it is able to specifically activate RAR-RXR heterodimers and not RXR-RXR homodimers (222). Presumably, both isomers of 4-oxo-RA can be synthesized from their respective RA isomers via a cytochrome P-450-linked oxidase, such as CYP26. Another potential source of 4-oxo-RA is 4-oxo-retinaldehyde, and this retinoid has been identified in early Xenopus embryos (12). Surprisingly, 4-oxo-retinaldehyde itself can activate the RAR, and it is the major bioactive retinoid in Xenopus (12). Another transactivator of RAR is 4-oxo-retinol, a compound present in both Xenopus and the mouse F9 teratocarcinoma cell line (12). The existence of such active ketone derivatives of the retinoids suggests the potential importance of cytochrome P-450-linked oxidases in activating retinoids, in addition to their role in retinoid catabolism (see sect. IVF).

In embryonic mouse (90) and human (119), retinol is present at micromolar concentrations, whereas retinaldehyde levels are barely detectable. This contrasts with several cold-blooded organisms including zebrafish (29) and Xenopus (7), in which high levels of retinaldehyde are present, bound via the labile Schiff-base linkage to vitellogenin. High retinaldehyde concentrations in the zebrafish embryo place even greater emphasis on the retinaldehyde dehydrogenases as the determinant of RA synthesis (29). The substrates all-trans-retinaldehyde, all-trans-retinol, and didehydro-retinol all appear to be localized to specific regions of the early Xenopus embryo (118), a distribution that is likely to influence the distribution of RA and didehydro-RA. We have found this to be the case for the retinaldehyde distribution in the zebrafish trunk (170). The distribution of the retinyl esters has not been investigated in the developing embryo, but both retinyl acetate and retinyl palmitate have been identified in cultured cells from the chick retina (266).

A number of pathways of RA catabolism have been described, which may be tissue or species specific (6, 70, 113, 118, 254). These routes include oxidation, isomerization, and formation of glucuronides and taurine conjugates. Recently, a new member of the cytochrome P-450 family, CYP26 (or P-450RAI), has been identified as an enzyme in the embryo that specifically mediates RA oxidation (68), and it is presumed to mediate RA catabolism. CYP26 was first identified in the adult zebrafish as a RA-inducible catabolic enzyme (301) and was later cloned in mouse and human cells by several laboratories (230, 300). Its predominant metabolites have been described as either 4-hydroxy-RA, 18-oxo-RA, and 4-oxo-RA (230, 300), or as 5,8-epoxy-RA (68). The CYP26 promoter contains a RA response element; however, RA exposure of the mouse embryo induces expression only in a limited region (68), indicating the existence of other transcriptional regulators for this gene. Its normal expression pattern in the embryo is suggestive of a function of regulating RA distribution. In the mouse (68), at embryonic day 7.25, it is present in a posterior to anterior gradient in the mesoderm. High expression at the posterior end continues through embryonic day 8.5 to be present in the posterior neural plate and mesoderm of the hindgut and tailbud at embryonic days 9.5-10.5. Localized areas of anterior expression also exist, with high levels in the neural crest cells of the hindbrain which migrate to the cranial ganglia. In the CNS, expression is also present in the otic vesicle and eye (68). As described above, the presence of CYP26 in the retina is particularly revealing since it sits as a midline stripe subdividing the two RA synthetic enzymes in the dorsal and ventral (181). This suggests a possible role for CYP26 in boundary formation, acting as a perimeter between regions of high RA synthesis. A function, for CYP26, of boundary delineation in the Xenopus hindbrain is also suggested by Hollemann et al. (88). The existence of catabolic enzymes in the mouse hindbrain in addition to CYP26 has also been reported (309). It is likely that the patterning of the catabolic enzymes will be just as important as that of the synthetic enzymes for determining the sites of RA's action.

The retinoid binding proteins are likely to play a role in the regulation of retinoid metabolism, and they are widely expressed in the developing embryo (40, 51, 148, 219, 241, 242). Generally, they act as cytoplasmic carriers for the lipophilic retinoids, which they shuttle between both subcellular compartments and metabolic enzymes. They are not, however, absolutely essential for the embryo, because the loss of function of either CRBP-I or CRABP-I and -II has little effect on normal development (127). It has been suggested that their importance may become more evident under circumstances of low dietary vitamin A supply. It is also likely that alternative retinoid binding proteins exist (310).

CRBP-I is believed to promote RA synthesis by presenting retinol to the retinol dehydrogenase (200). After oxidation to retinaldehyde, this substrate can again bind to CRBP and is available to the retinaldehyde dehydrogenase (216). Apo-CRBP-I is also known to promote retinyl ester hydrolysis, leading to the release of retinol (200), which is then accessible to the retinaldehyde dehydrogenases.

CRABP-I is thought to promote the breakdown of RA (14) because the catabolic enzyme prefers holo-CRABP-I as a substrate (64). The embryonic distributions of CRABP-I is generally complementary to that of CRBP-I, possibly reflecting its contrasting function. Embryonic regions sensitive to the teratogenic effects of retinoids, including the developing neural crest and hindbrain, express high levels of CRABP-I, which led to the suggestion that CRABP-I serves a protective role in RA vulnerable tissues (40, 242, 287), consistent with a role in RA buffering and catabolism. In contrast to CRABP-I, CRABP-II has been shown to be associated with cells that synthesize RA in the adult uterus and testes, and it may play a role in RA synthesis or secretion (16, 320).

RA can function as an embryonic morphogen when synthesized by discrete organizer regions to create gradients of transcriptional activation. This is likely to occur in several regions of the embryo including the early eye anlage (55). The observations reviewed here demonstrate another mode of patterning in the form of zones of differing RA concentration. These territories are created by the ordered patterning of RA synthetic and catabolic enzymes in the embryo, and they have been observed in several regions during development, including the early heart (194) and the retina at later stages of embryonic development (181). A sharp drop off in RA levels occurs also between the spinal cord and the hindbrain in the embryonic mouse (175) and chick (156), and it was noted that gradients are not evident in the RA reporter mice (236). It appears that the zonal patterning of RA levels may be an important form of organization in addition to RA gradients. Several regions of retinaldehyde dehydrogenase expression show exceptionally high levels of enzyme activity, including the spinal cord and the retina (54, 175). The resulting RA concentration in the ventral retina probably exceeds 1 µM, far beyond the concentrations necessary for the activation of the RA receptors. Because the ventral retina is bounded by the catabolic enzyme, this high concentration cannot serve to create a continuous RA gradient across the entire retina. One possible explanation for the high retinaldehyde dehydrogenase levels in neurons may relate to the observations that high enzyme levels are not restricted to the cell bodies but extend also into the axons. Very high AHD2 concentrations are present in the retinal axons (180) and in the dopaminergic axons innervating the corpus striatum (176), and RALDH2 is found in axons of the motoneurons projecting from the embryonic spinal cord (10) Axons from the retina, the ventral tegmentum, and the spinal cord innervate regions that do not contain synthetic enzyme but do express RA receptors. If the axon terminals represent sources of RA, then high enzyme activities would be required in the soma region, to provide amounts for the transport through the thin axons which are sufficient for target activation. Territories of high RA may also represent RA sources for neurons whose processes make contact or pass through these regions, because RA diffuses readily through cell membranes. Fibers innervating the spinal cord motoneurons, or passing by in their vicinity, are exposed to the very high RA levels present here. Cells of the neural crest, which require RA (151) but do not express RALDH2 (10), migrate through mesenchyme which expresses high levels of RALDH2 (206). Such exposure may be a necessary part of their maturation. Cell types that do not express retinaldehyde dehydrogenases but that respond to external sources of RA may have to be efficient at RA capture. This may be a function of CRABP-I, since it is almost exclusively expressed in cells that respond to RA but that do not themselves synthesize RA. Examples include the neural crest (104), cerebellum (311), and interneurons of the spinal cord (253). The normal response of these tissues to low concentrations of RA might explain why these regions are so sensitive to RA teratogenicity (9, 287); the exposure to abnormally high RA levels will disturb their normal sequence of gene expression. The axons of several neurons are known to express CRABP-I (147, 154, 253), and the binding proteins may assist in the capture of RA, allowing for its transport back to the cell body. The polarization of neurons into soma and far-reaching axonal projections makes it likely that axonal transport plays a role in the localized capture and release of RA. There can be little doubt that the developing CNS will be an interesting area of investigation for the interactions between retinoid metabolism, signaling, and patterning of gene expression.

	V. RETINOID RECEPTORS/FUNCTION

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The cloning and characterization of the retinoid receptors represent a landmark in our understanding of the physiology of the retinoids. The discovery was a direct consequence of basic work in the field of steroid and thyroid hormone receptors. A characteristic of these receptors is their modular structure with six different domains (A to F) that fulfill different functions (21, 160). The DNA-binding domain or DBD, also known as the C domain, is very highly conserved between these different receptors. Retinoid receptor cloning was possible because of the common 24-nucleotide probe within this C region used to identify new orphan receptors (71, 221). These approaches permitted the characterization of three genes (, , and ) for RAR and three genes for RXR (159). The latter class of receptors specifically bind 9-cis-RA, whereas RAR bind both RA and 9-cis-RA (35, 137, 159).

Genes containing retinoic acid response elements (RARE) in their promoters are known to be involved in diverse and yet interconnected biological processes, such as embryogenesis, growth, and differentiation (35, 160, 161). The complexity of interactions becomes greater if one considers that RXR, in addition to forming homodimers (159), also form heterodimers with other receptors (135, 159, 166, 314), including RAR, the thyroid hormone receptor TR, the 1,25-dihydroxy-vitamin D₃ receptor VDR (314), the peroxisomal proliferator activated receptor PPAR (105, 108, 282), NGFI-B, NURR1 (220), and COUP-TF (106). A distinctive characteristic of the RXR is their ability to interact with other nuclear receptors of the superfamily resulting in their binding to their respective DNA response elements in the promoters of different target genes (107). Gene transcription responses are RXR partner receptor dependent as well as dependent on the presence of the ligand of their heterodimeric partner, e.g., vitamin D₃ for the VDR/RXR combination, thyroid hormone for the TR/RXR combination, and so on. It is therefore easy to conceptualize how RXR may control a complex network of hormone-dependent pathways. Complexity increases if one also considers that there are some 18 isoforms of the RAR and probably just as many RXR formed through the use of different promoters or alternative splicing (134).

In addition to the activation function AF-1 in the NH₂-terminal A/B regions of the receptors, the use of various reporter gene assays has made it possible to identify a transcriptional activation function (AF-2), which overlaps the ligand binding domain (LBD) in the E region of the RAR and RXR. RAR AF-2 are activated similarly by all-trans- and 9-cis-RA and their 3,4-didehydroderivatives (Fig. 1), whereas RXR AF-2 are only efficiently activated by 9-cis-RA and by 9-cis-3,4-ddRA (see Ref. 134 and references therein).

A third level of complexity accrues from interactive elements on different promoters. For instance, response elements containing direct repeats (DR) of the canonical sequence AGGTCA with a spacer of two nucleotides (DR2) or five (DR5) are known to occur in the homeobox b1 (169, 269) and RAR2 genes. In addition, the actual sequence of the direct repeats and the types of flanking bases appear to be important determinants for RAR and RXR binding efficiencies (157). Moreover, interactions with other proteins, which may either function as transcriptional repressors (73, 89, 122, 316) or ligand-dependent activators (20, 23, 97), such as the cAMP response element binding protein (CBP), have also been reported and may add another level of complexity as well as permit a variety of interactive pathways to regulate specific gene expression.

These considerations suggest different orders of interactions that may lead to diverse biological outcomes. We suggest that effects of interactions between RXR-RAR heterodimers and target gene promoters are the most immediate and would be the first to respond to retinoid depletion or excess. These reactions would acquire the characteristic immediate response, if the RARE is located in the promoter of the target gene itself. This is the case for the RAR2 gene, which is therefore a "first-order dependence gene." Other first-order dependence genes would be the RAR2 and 2 and all the genes that contain a RARE in their promoters. Some of these are listed in Table 1.

First-order dependence would also be observed with ligands activating RAR-RAR or RXR-RXR homodimers, such as for 9-cis-RA in the activation of the CRBPII gene (Table 1). RXR cognate receptors other than RAR, such as VDR, TR (17, 107, 166, 318), COUP-TF (106), and PPAR (282), would mediate second-order types of retinoid responses, in that their responses, although possibly dependent on 9-cis-RA (188), are also dependent on other hormonal ligands such as vitamin D₃, thyroxine (314), as well as xenobiotic agents.

We suggest that genes belonging to the "second-order retinoid dependence" are probably much more numerous than those of first-order dependence. These genes control most gene activation processes dependent on thyroid hormone, vitamin D, and other important hormonal and xenobiotic ligands, which also depend on heterodimer formation with RXR. We call "third-order retinoid-dependent genes" all those genes that would be activated as the result of secondary transcriptional events. An example of this type of dependence is genes that depend on AP-1 complexes. It is well known that transcriptional trans-repression occurs at the AP-1 site concomitant with RA-mediated gene activation on various RARE, especially when RA is in excess. Genes containing 12-O-tetradecanoylphorbol-13-acetate (TPA) responsive elements (TRE), such as the stromelysin or the collagenase genes, are negatively regulated by RA. This negative regulation was thought to be due to binding of the AP-1 Jun-Fos protein to the RAR, thus removing them from interactions with the TRE. More recent results indicate that trans-repression is the result of limiting concentrations of the cAMP response element binding protein (CREB) and the protein that binds to CREB or CBP. This CBP apparently binds with high affinity to CREB as well as to RAR, estrogen receptor (ER), and possibly a variety of other receptors (97). Whereas the binding and transcriptional activation by the CREB homodimer is independent of ligands, CBP binding to RAR and other hormone receptors and consequent transcriptional activation is ligand dependent (97). Because CBP is present in limiting amounts, AP-1 trans-repression normally occurs when RAR or the ER concentrations are increased. When CBP is nonlimiting, trans-repression is not observed. Therefore, the concentration of CBP and of other ligand-dependent coactivators, as well as that of corepressors, add another level of transcriptional control. Recent reviews on the important aspects of structural configuration and space-filling models (159) of the retinoid receptors and on the genetic knockout (99) of their genes have recently been published.

Data from in situ hybridization studies show that different RAR and RXR subtypes are widely expressed during embryogenesis and that each subtype has its own individual expression patterns that may or may not overlap with the other subtypes. Each retinoid receptor subtype can give rise to different isoforms, each with their own unique expression patterns during embryogenesis (62). Retinoid nuclear receptors of different types appear to be expressed along the entire anterior/posterior (A/P) axis in the CNS. The data suggest that individual receptor subtypes have specific and probably multiple functions. The RAR subtype is expressed in a general fashion during murine embryogenesis, whereas RAR and RAR are more restricted (52, 183, 239-241). In the hindbrain, RAR has a rostral limit of expression at the rhombomere 6/7 boundary (149), and RAR is expressed in the open neural tube before the fusion of neural folds (239). Among the RXR, murine RXR is expressed in a general fashion, but RXR and RXR have more restricted patterns during embryogenesis (48, 158).

The patterned expression or suppression of developmental genes (e.g., the homeobox b1 gene) has been demonstrated to be strictly retinoid responsive, in specific rhombomeres of the mouse hindbrain (168). In fact, RARE have been demonstrated both 5' as well as 3' of the gene with the 5'-RARE functioning as a suppressor and the 3'-RARE as an inducer of transcriptional activity for the homeobox b1 gene (38, 168, 269). The developmental time dependency of the function of the two RARE permits and controls the switching on and off of the homeobox ₁-gene expression and termination of expression at the border of different rhombomeres, possibly controlling segment identity. It is also evident that a highly ordered retinoid delivery system must be operative to ensure the regulated series of developmental events. Availability of excess retinoids or their deficiency may lead to teratogenesis and/or resorption of the embryo.

	VI. RETINOID RECEPTOR KNOCKOUT MUTANTS

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Much effort has been applied over the past few years to the identification of the specific functions of the different retinoid receptors during embryogenesis. One strategy has been to evaluate the phenotype of mice lacking the gene for a retinoid receptor or receptor subtype. Although the different receptor isoforms have unique distributions during development, knocking out one specific isoform revealed a surprising redundancy between members of each receptor subtype. Mice homozygous for either RAR1, RAR2, or RAR2 mutation were viable and did not display phenotypic abnormalities (99). Since these initial studies, a stepwise progression of mutant mice from single isoform retinoid receptor mutants to RAR subtype mutants to RAR/RXR compound mutant mice have been created to examine the in vivo function of these receptors (Table 2).

Developmental malformations can arise as a result of knocking out the entire gene of a single retinoid receptor. For example, homozygous RAR mutant mice, which express no RAR isoform transcripts, exhibited early postnatal lethality and testis degeneration (145). Of the RAR single-type null mutations, RAR knockout mice showed malformations (138). RAR mutant mice exhibited several abnormalities previously associated with vitamin A deficiency, including homeotic transformations of the cervical vertebra and occipital region of the skull, fusion of the first and second ribs, and irregularities of the tracheal rings, among others (138).

Because of their interaction with several members of the steroid/thyroid hormone receptor superfamily, the absence of a RXR would be expected to have more serious consequences during embryogenesis. One such gene knockout, RXR, resulted in morphologically normal mice, with the exception that males were sterile (101). Homozygous RXR mutant mice died between gestation days 13.5 and 16.5 (271). The embryonic lethality in RXR-deficient mice was due to hypoplastic development of the ventricular chambers of the heart, which resulted in a very thin ventricular wall and defects in ventricular septation. RXR null mutants showed abnormalities of the eye (e.g., shortening of the ventral retina) as also observed in vitamin A deficiency (Table 2).

None of the phenotypes resulting from single knockouts, however, was expected on the basis of expression patterns of retinoid nuclear receptors during embryogenesis. Although some specific aberrations have been found to be associated with the lack of certain nuclear receptor subtypes, these studies seem to suggest a high degree of functional redundancy among these receptors. The results of single RAR and RXR null mutants suggest that embryogenesis is well protected by a system that apparently allows most or all functions of an absent receptor to be substituted by another.

C.  Double/Compound Mutants

1.  RAR

Unlike RAR single mutants, RAR double or compound mutants died either in utero or shortly after birth (139, 140, 146, 184). Histological and anatomical analyses of these transgenic mice revealed many defects characteristic of fetal vitamin A deficiency (Table 2). Multiple eye abnormalities were found in various RAR double mutant fetuses that were similar to those previously seen in vitamin A-deficient fetuses. A majority of these abnormalities recapitulated those observed in the fetal vitamin A-deficient syndrome, initially described more than 40 years ago (Table 2) (305). A number of additional malformations not described in vitamin A deficiency studies, however, were also observed (140, 184). These findings probably reflect the difficulty of achieving severe vitamin A deficiency by dietary deprivation (99). Taken collectively, these results demonstrate that RAR are essential for vertebrate development and that, most likely, retinoic acid is the active retinoid, required at several stages of the development of numerous tissues and organs (Table 2). In contrast to the apparent necessary role for RAR in transducing the RA signal, CRABPI and CRABPII are probably not critical to these processes, since CRABPI/CRABPII double null mutant mice were found viable and morphologically normal (127).

Double or triple mutations of RXR would be expected to reproduce the defects associated with inactivations of RAR/RXR heterodimers or, at least, produce defects similar to the RAR double mutant defects. Strikingly, RXR / RXR / double and RXR +/ RXR / RXR / triple mutant phenotypes were viable, displaying no obvious congenital or even postnatal abnormalities, except a marked growth deficiency and male sterility due to loss of function of RXR (121). Therefore, it appears that one copy of RXR is sufficient to perform most of the functions of the RXR.

Kastner and co-workers (99, 100) performed a massive undertaking to determine the functional significance of RXR/RAR heterodimeric signaling in embryogenesis. These investigators created compound mutant fetuses bearing null alleles in one RXR (, , or ) and one RAR (, , or ) subtype or isoform gene (100). The data presented show synergy between the effects of RXR and RAR mutations, but no such synergy was observed when RXR or RXR mutations were combined with RAR mutations. It is also noteworthy that all defects of fetal vitamin A deficiency are recapitulated by the various RXR/RAR compound mutations (Table 2). Some of the abnormalities were specific to one type of RXR/RAR mutant combination, whereas others were seen in several types of RXR/RAR double mutants. As an example of specificity, the study of RAR double mutants has suggested that RAR and RAR2, as well as RAR and RAR, are functionally redundant for the formation of the aorticopulmonary septum (184). In all RXR/RAR double mutants, a complete absence of this septum was observed. This suggests little functional redundancy between the RAR subtypes, since RAR can never be functionally replaced by either RAR or RAR2 in a RXR mutant background. The case for multiplicity of function may indicate that more than one RXR/RAR pair is normally involved in the underlying developmental process, which may require the expression of several RA target genes. These data confirm the role of RXR/RAR heterodimers as the functional units that transduce the retinoid signal for a large number of RA-dependent processes. These results further implicate RXR as the main RXR in the developmental functions of RAR. However, it is noted that a tight functional specificity of RXR for heterodimerization with RAR is unlikely, because many RAR-dependent developmental processes proceed normally in mutants that express RXR as the only RXR (121).

Mouse embryos lacking RXR have normal limbs and display resistance to limb malformations normally induced by teratogenic RA exposure (272). RA treatments that cause limb defects in 100% of wild-type embryos failed to elicit malformations in RXR null homozygotes, implicating RXR as a component in the teratogenic process in the limbs. Furthermore, heterozygous embryos are intermediate in their sensitivity to RA, suggesting the importance of RXR gene dosage in limb teratogenesis. These investigators, however, found that expression of the RA-inducible gene RAR2 was equivalent between wild-type and homozygous RXR embryos after RA treatment. The spatial expression of sonic hedgehog (Shh) and Hoxd12 was also similar for both wild-type and RXR embryos, following RA treatment. Hoxd12 expression, however, was elevated in RXR embryos. These observations indicate that transcriptional processes, which are inappropriately influenced in the mouse limb by exogenous RA, require RXR for their execution.

Results from double mutant mice provide additional information for the role of RA in limb patterning (100). Because both RAR and RAR transcripts are uniformly expressed in the early stage mouse limb bud (51), it was surprising that neither RAR nor RAR single knockout mice produced limb malformations (138, 145). These observations, however, suggested a functional redundancy between these two receptors. This may well be the case since limbs from RAR/RAR double mutants consistently exhibited malformations, including size reduction of the scapula, perforated scapula, radius agenesis, and abnormal digit number (140). Many of these limb defects appear to be fairly restricted to the forelimb skeleton and may reflect a requirement of RA to generate the proper amount of limb mesenchyme, since a deficit of mesenchyme leads to preferential loss of anterior skeletal elements in frog hindlimbs and preferential loss of posterior skeletal elements in salamander hindlimbs (3). The defects do not appear to result from an early zone of polarizing activity (ZPA) defect because limbs displayed a clear A/P asymmetry. The investigators suggest this does not exclude a role for RA in normal A/P limb patterning, since RAR transcripts are expressed in a region that overlaps with the ZPA and appear to be unaffected in the limbs of RAR/RAR double mutants (140). The observations from the limb defects in RAR/RAR double mutants suggest that either RA plays different roles in forelimb and hindlimb development, or the observation is related to events occurring at different developmental time periods for the two limbs. It has also been suggested that inactivation of all three RAR might result in more dramatic effects on limb patterning (99).

RAR and occasionally RAR single mutants show homeotic transformations and malformations of vertebrae (138, 140). These studies establish that RA plays an important role in patterning of the main body A/P axis. Penetrance and expressivity of cervical anterior transformations observed in RAR null mice increased in a graded manner, with subsequent loss of RAR1 and RAR2 isoforms from the RAR background (140). Furthermore, concomitant inactivation of all RAR and - isoforms resulted in severe degeneration of the cervical vertebrae (140). RAR2 may also be involved in axial patterning, because RAR2 (but not RAR) double mutants displayed a high frequency of anterior transformations of the sixth and seventh vertebrae (140). Cervical region patterning has also been examined after inactivation of one RXR allele within several RAR mutant backgrounds (100). Of these, the RXR +/, RAR +/, as well as RXR +/ RAR +/ newborns exhibited a high frequency of defects in the cervical region. The nature of the vertebral abnormalities in RAR single and double mutants as well as RXR/RAR mutant mice is of significance in view of the interactions between RA and Hox gene signaling. In the somitic mesoderm of the future vertebral column, each vertebral level has a specific Hox gene specification