Retinoic acid synthesis for the developing telencephalon.

The small lipid retinoic acid is known to promote neuronal differentiation in vitro and to act as a teratogen in the embryonic brain, but very little is known about the natural role of endogenously synthesized retinoic acid in forebrain development. Retinoic acid is synthesized mainly by three retinaldehyde dehydrogenases. We show here where the retinaldehyde dehydrogenases for the developing telencephalon are expressed and how their expression patterns change over developmental time. Retinoic acid diffusing from the retinaldehyde dehydrogenase sites is likely to influence the early telencephalon before the beginning of neurogenesis, as well as differentiation and radial migration of neurons into the cerebral cortex. Because of its diffusible character, retinoic acid represents a unique tool for the coordination of growth processes over an intermediate distance range in the developing telencephalon.

Retinoids in embryonal development.

The key role of vitamin A in embryonal development is reviewed. Special emphasis is given to the physiological action of retinoids, as evident from the retinoid ligand knockout models. Retinoid metabolism in embryonic tissues and teratogenic consequences of retinoid administration at high doses are presented. Physiological and pharmacological actions of retinoids are outlined and explained on the basis of their interactions as ligands of the nuclear retinoid receptors. Immediate target genes and the retinoid response elements of their promoters are summarized. The fundamental role of homeobox genes in embryonal development and the actions of retinoids on their expression are discussed. The similarity of the effects of retinoid ligand knockouts to effects of compound retinoid receptor knockouts on embryogenesis is presented. Although much remains to be clarified, the emerging landscape offers exciting views for future research.

A retinoic acid synthesizing enzyme in ventral retina and telencephalon of the embryonic mouse.

Most retinoic acid (RA) in the embryonic mouse is generated by three retinaldehyde dehydrogenases (RALDHs). RALDH1 (also called E1, AHD2 or ALDH1) is expressed in the dorsal retina, and RALDH2 (V2, ALDH11) generates most RA in the embryonic trunk. The third one, RALDH3 (V1), synthesizes the bulk of RA in the head of the early embryo. We show here that RALDH3 is a mouse homologue to ALDH6, an aldehyde dehydrogenase cloned from adult human salivary gland (Hsu, L.C., Chang, W.-C., Hiraoka, L., Hsien, C.-L., 1994. Molecular cloning, genomic organization, and chromosomal localization of an additional human aldehyde dehydrogenase gene, ALDH6. Genomics 24, 333-341), which was recently reported to act as a RALDH (Yoshida, A., Rzhetsky, A., Hsu, L.C., Chang, C., 1998. Human aldehyde dehydrogenase gene family. Eur. J. Biochem. 251, 549-557). RALDH3 expression begins in the surface ectoderm over the optic recess. In rapidly changing expression patterns it labels the appearance of several ectodermal structures: it marks the formation of the lens and the olfactory organ from ectodermal placodes, and it delineates the beginning eyelid field. Within the optic vesicle, RALDH3 is expressed in the ventral retina and the dorsal pigment epithelium. In the telencephalon, RALDH3 is expressed at high levels in the lateral part of the ganglionic eminence. From here it extends via the piriform cortex into the lower part of the septum. Of the three RALDHs, RALDH3 shows the strongest predilection for epithelia.

Retinoic acid in the formation of the dorsoventral retina and its central projections.

Dynamic expression patterns of four retinoid-metabolizing enzymes create rapidly changing retinoic acid (RA) patterns in the emerging eye anlage of the mouse. First, a RA-rich ventral zone is set up, then a RA-poor dorsal zone, and finally a tripartite organization consisting of dorsal and ventral RA-rich zones separated by a horizontal RA-poor stripe. This subdivision of the retina into three RA concentration zones is directly visible as beta-galactosidase labeling patterns in retinas of RA-reporter mice. Because the axons of retinal ganglion cells transport the reporter product anterogradely, the central projections from dorsal and ventral retina can be visualized as two heavily labeled axon bundles. Comparisons of the axonal labeling with physiologic recordings of visual topography in the adult mouse show that the labeled axons represent the upper and the lower visual fields. The RA-poor stripe develops into a broad horizontal zone of higher visual acuity. Comparisons of the retina labeling with eye-muscle insertions show that the axis of the RA pattern lines up with the dorsoventral axis of the oculomotor system. These observations indicate that the dorsoventral axis of the embryonic eye anlage determines the functional coordinates of both vision and eye movements in the adult.

Regulation of retinoic acid signaling in the embryonic nervous system: a master differentiation factor.

This review describes some of the properties of retinoic acid (RA) in its functions as a locally synthesized differentiation factor for the developing nervous system. The emphasis is on the characterization of the metabolic enzymes that synthesize and inactivate RA, and which determine local RA concentrations. These enzymes create regions of autocrine and paracrine RA signaling in the embryo. One mechanism by which RA can act as a differentiation agent is through the induction of growth factors and their receptors. Induction of growth factor receptors in neural progenitor cells can lead to growth factor dependency, and the consequent developmental fate of the cell will depend on the local availability of growth factors. Because RA activates the early events of cell differentiation, which then induce context-specific differentiation programs, RA may be called a master differentiation factor.

Dorsal and ventral rentinoic territories defined by retinoic acid synthesis, break-down and nuclear receptor expression.

Determination of the dorso-ventral dimension of the vertebrate retina is known to involve retinoic acid (RA), in that high RA activates expression of a ventral retinaldehyde dehydrogenase and low RA of a dorsal dehydrogenase. Here we show that in the early eye vesicle of the mouse embryo, expression of the dorsal dehydrogenase is preceded by, and transiently overlaps with, the RA-degrading oxidase creating a trough between very high ventral and moderately high dorsal RA levels. Most of the RA receptors are expressed uniformly throughout the retina except for the RA-sensitive RARbeta, which is down-regulated in the CYP26 stripe. The orphan receptor COUP-TFII, which modulates RA responses, colocalizes with the dorsal dehydrogenase. The organization of the embryonic vertebrate retina into dorsal ventral territories divided by a horizontal boundary has parallels to the division of the Drosophila eye disc into dorsal, equatorial and ventral zones, indicating that the similarities in eye morphogenesis extend beyond single molecules to topographical patterns.

Sagittal band expression of COUP-TF2 gene in the developing cerebellum.

In the developing cerebellum, the medio-lateral compartmentalization of the adult cerebellum is preceded by the transient expression of factors which divide the cortex into similar parasagittal stripes. Here we report that COUP-TF2, an orphan member of the nuclear receptor family which suppresses RA actions by forming heterodimers with RXR, shows a pattern of sagittal bands in developing mouse cerebellum. The band pattern changes according to the developmental stage. At embryonic day 13 it is expressed in the lateral half of the cerebellum, but at later stages the expression is divided into several parasagittal bands. By postnatal day 5 the COUP-TF2 expression substantially decreases to low, but detectable, levels.

Dorsal and ventral retinal territories defined by retinoic acid synthesis, break-down and nuclear receptor expression.

Determination of the dorso-ventral dimension of the vertebrate retina is known to involve retinoic acid (RA), in that high RA activates expression of a ventral retinaldehyde dehydrogenase and low RA of a dorsal dehydrogenase. Here we show that in the early eye vesicle of the mouse embryo, expression of the dorsal dehydrogenase is preceded by, and transiently overlaps with, the RA-degrading oxidase CYP26. Subsequently in the embryonic retina, CYP26 forms a narrow horizontal boundary between the dorsal and ventral dehydrogenases, creating a trough between very high ventral and moderately high dorsal RA levels. Most of the RA receptors are expressed uniformly throughout the retina except for the RA-sensitive RARbeta, which is down-regulated in the CYP26 stripe. The orphan receptor COUP-TFII, which modulates RA responses, colocalizes with the dorsal dehydrogenase. The organization of the embryonic vertebrate retina into dorsal and ventral territories divided by a horizontal boundary has parallels to the division of the Drosophila eye disc into dorsal, equatorial and ventral zones, indicating that the similarities in eye morphogenesis extend beyond single molecules to topographical patterns.

Postnatal effects of retinoic acid on cerebellar development.

Retinoic acid(RA) is a potent teratogen to which the early CNS is known to be highly sensitive. However, very little is know about the postnatal effects of RA. The cerebellum is a candidate for postnatal RA toxicity, as it develops late and exhibits temporal patterns of RA synthesis that are synchronized with developmental stages. Northern blotting shows that the cerebellum expresses receptors for RA, predominantly of the RXR group activated by 9-cis RA. To determine whether development can be disrupted by RA excess, newborn rats were injected with RA at a time when endogenous RA levels are normally low. Histological examinations at postnatal day 14 revealed loss of a subpopulation of proliferating granule cells, and adult cerebella showed clusters of ectopic granule cells located in the molecular layer. Thus, although the granule cells may normally be regulated by endogenous RA, they are sensitive to abnormally high RA concentrations.

Retinoid-binding proteins in the cerebellum and choroid plexus and their relationship to regionalized retinoic acid synthesis and degradation.

The expression of cellular retinoic-acid-binding protein (CRABP) and cellular retinol-binding protein (CRBP), as well as their relationship to retinoic acid (RA) synthesis and degradation were examined in the developing mouse cerebellum and choroid plexus of the fourth ventricle. The choroid plexus, which expresses the RA-synthesizing retinaldehyde dehydrogenase RALDH-2, is likely to represent a diffusion source of RA for the closely apposed cerebellum, regulating its development. We found CRBP to be expressed in the choroid plexus and, in an in-vitro assay, addition of recombinant CRBP to RALDH-2 increased RA synthesis from retinaldehyde, with the amount of increase depending on the CRBP/retinaldehyde ratio. A technique that characterizes RA-binding proteins according to their isoelectric point showed both CRABP I and CRABP II to be present in the cerebellum and P19 cells, and only CRABP II to be present in the choroid plexus. With this technique, CRABP I could also be detected in the HL60 cell line. In addition to the two known acidic RA-binding proteins CRABP I and II, the cerebellum expressed a third RA-binding protein distinguishable by its neutral isoelectric point; the same binding protein was also detected in the olfactory bulb, kidney and testes. We used the RA-binding technique to follow the rate of elimination of bound RA from the cerebellum. A systemic injection of 0.3 micromols RA into postnatal day-1 mice was almost completely removed after 8 hours. These results suggest mechanisms by which the retinoid-binding protein may regulate the equilibrium of RA synthesis and catabolism in the cerebellum and choroid plexus.

Dynamic patterns of retinoic acid synthesis and response in the developing mammalian heart.

Retinoic acid (RA) has been implicated in cardiac morphogenesis by its teratogenic effects on the heart, although its role in normal cardiogenesis remains unknown. To define the parameters of RA action in cardiac morphogenesis, we analyzed the patterns of ligand synthesis, response, and inactivation in the developing mouse heart. Activation of a lacZ transgene controlled by an RA response element (RARE) was compared to the localization of the retinaldehyde-oxidizing dehydrogenase RALDH2, the earliest RA synthetic enzyme in the mouse embryo, and to the expression of a gene encoding an RA-degrading enzyme (P450RA). We observed that RALDH2 localization and RA response were virtually superimposable throughout heart development. Initially, both RALDH2 and RARE-LacZ activity were restricted to the sinus venosa in unlooped hearts, but were high in the dorsal mesocardium, while P450RA expression was restricted to the endocardium. Later stages were characterized by a sequential, noncontiguous progression of RALDH2 accumulation and RA response, from the sinus venosa to atria, dorsal-medial conotruncus, aortic arches, and the epicardium. This dynamic pattern of RA response was a direct result of localized RALDH2, since hearts of cultured embryos were uniformly competent to respond to an exogenous RA challenge. These observations support a model in which the influence of endogenous RA on heart development depends upon localized presentation of the ligand, with only limited diffusion from the source of its synthesis.

A novel assay for retinoic acid catabolic enzymes shows high expression in the developing hindbrain.

We have employed a novel technique that determines the relative capacity of tissues to catabolize all-trans retinoic acid (RA) to a metabolite incapable of activating a RA reporter cell line. This assay uses the microsomal fraction of tissues from the developing mouse and detects a pathway which requires NADPH and is inhibitable by ketoconazole, suggesting that a cytochrome P450-dependent enzyme may be required. High catabolic activity was detected transiently in the developing cerebellum which peaked at postnatal day 2. The medulla oblongata was the only other CNS region with high catabolic capacity, its earlier expression peak, between embryonic days 16 and 17, likely reflecting its earlier maturation. In the CNS, the hindbrain is exceptional in its high expression of RA catabolic enzymes, suggesting a unique function for regulated RA levels in this region.

Aldehyde dehydrogenases in the generation of retinoic acid in the developing vertebrate: a central role of the eye.

In the developing vertebrate, retinoic acid is distributed in patterns that are highly regulated, both in the spatial and temporal domains. These patterns are generated by the localized expression of retinoic acid-synthesizing aldehyde dehydrogenases, which form the origins of retinoic acid-diffusion gradients in the surrounding tissues. The developing eye, known to be exceptionally vulnerable to vitamin A deficiency, is one of the retinoic acid-richest regions in the embryo. Several aldehyde dehydrogenases are expressed here, and they create a ventro-dorsal retinoic acid gradient in the embryonic retina. Aldehyde dehydrogenase expression persists in the mature eye and is stable, but the amount of retinoic acid synthesized is variable, depending on ambient light levels. This phenomenon is due to changing levels of the retinoic acid precursor retinaldehyde, which is released from illuminated rhodopsin, thus providing a mechanism by which light can directly influence gene expression. For arrestin mRNA, which is one of the factors known to be regulated by light, the light effect can be mimicked in the dark by injection of retinoic acid. The light-induced release of retinaldehyde from rhodopsin, which occurs only in vertebrate but not invertebrate photoreceptors, may have accelerated the rapid evolution of retinoic acid-mediated transcriptional regulation at the transition from invertebrates to vertebrates, and it may explain the prominent role of retinoic acid in the eye.

Retinoic acid synthesis in the developing chick retina.

The transcriptional activator retinoic acid (RA) has been shown to influence the early patterning of the vertebrate eye. Models for the establishment of the retinofugal projection postulate gradients of cell-surface markers across the retinal surface that are expressed by ganglion cells and mediate the correct connection of fibers within central target fields. Spatial asymmetries of RA and RA-producing enzymes, as have been found in the eyes of mice and zebrafish, could induce the required asymmetry in gene expression. Here we exploited the large size of the retina of the embryonic chick to analyze the spatial and temporal characteristics of the RA system by HPLC in combination with a reporter cell assay. As in other embryonic vertebrates, the chick retina was found to contain different RA-generating enzymes segregated along the dorsoventral axis. The major RA isomer in both dorsal and ventral retina was all-trans RA, and no 9-cis RA could be detected. This excludes a difference in production of these two isomers as an explanation for the expression of different RA-generating enzymes. At developmental stages embryonic days (E) 4 and 5, the ventral retina contained higher all-trans RA levels than the dorsal retina. After E8, however, the difference disappeared, and in embryos at E9 and older the RA concentration was slightly higher in dorsal than ventral retina.

A ventrodorsal GABA gradient in the embryonic retina prior to expression of glutamate decarboxylase.

GABA is known to function as a neurotransmitter in the mature nervous system, and in immature neurons it has been linked to neurotrophic actions. While most GABA is generated by glutamate decarboxylase (GAD), an alternative synthetic pathway is known to originate from putrescine, which is converted via gamma-aminobutyraldehyde in an aldehyde-dehydrogenase-requiring step to GABA. In a search for the role of two aldehyde dehydrogenases expressed in segregated compartments along the dorsoventral axis of the developing retina, we assayed dorsal and ventral retina fractions of the mouse for GABA by high performance liquid chromatography. We found GABA to be present in the embryonic retina, long before expression of GAD, and ventral GABA levels exceeded dorsal levels by more than three-fold. Postnatally, when GAD became detectable, overall GABA levels increased, and the ventrodorsal concentration difference disappeared. Our observations indicate that prior to the formation of synapses the embryonic retina contains a ventrodorsal GABA gradient generated by an alternate synthetic pathway.

A sensitive bioassay for enzymes that synthesize retinoic acid.

Retinoic acid (RA) is a potent regulator of gene transcription and it plays a pivotal role in neural development. As the compound is active at nanomolar concentrations, standard RA detection methods based on high-pressure liquid chromatography (HPLC) are poorly suited for neurodevelopmental questions and single RA measurements require pooling of tissues from very large numbers of embryos. An alternative approach is to determine the potential for RA synthesis by assaying for the enzymes that catalyze the last step of RA synthesis, the irreversible oxidation of retinaldehyde to RA. In a quantitative comparison of retinaldehyde dehydrogenase levels with endogenous RA levels, we found a good concordance between the two parameters. For the detection of retinaldehyde dehydrogenases we have developed an assay which involves analyses of protein fractions, separated by isoelectric focusing (IEF), with a RA responsive cell line; operationally, this technique is a zymography bioassay. This method is exquisitely sensitive: tissue volumes as small as a sugar grain can be assayed, and it can be used on all species. Separation by IEF allows in a single run the identification and relative quantitation of several enzyme isoforms.

Retinoic aid increases arrestin mRNA levels in the mouse retina.

Arrestin, which plays a role in the termination of the visual transduction cascade, is one of several photoreceptor proteins whose mRNA levels are increased by light. Retinoic acid, a by-product of photoreceptor signaling and a potent modulator of hormonal transcription control, is one candidate for regulating the arrestin mRNA levels. Here we show that retinoic acid, injected intraperitoneally into dark-adapted mice, increases the arrestin mRNA levels and mimics the effect of light. Injection of 1 mumol of retinoic acid produces a maximal increase in arrestin mRNA levels. The mRNA level reaches a maximum 3 h after injection and slowly declines thereafter. The observations suggest that retinoic acid may mediate the increase in arrestin mRNA produced by light.

Restricted expression and retinoic acid-induced downregulation of the retinaldehyde dehydrogenase type 2 (RALDH-2) gene during mouse development.

Retinaldehyde dehydrogenase type 2 (RALDH-2) was identified as a major retinoic acid generating enzyme in the early embryo. Here we report the expression domains of the RALDH-2 gene during mouse embryogenesis, which are likely to indicate regions of endogenous retinoic acid (RA) synthesis. During early gastrulation, RALDH-2 is expressed in the mesoderm adjacent to the node and primitive streak. At the headfold stage, mesodermal expression is restricted to posterior regions up to the base of the headfolds. Later, RALDH-2 is transiently expressed in the undifferentiated somites and the optic vesicles, and more persistently along the lateral walls of the intraembryonic coelom and around the hindgut diverticulum. The RALDH-2 expression domains in differentiating limbs, which include presumptive interdigital regions, coincide with, but slightly precede, those of the RA-inducible RAR beta gene. The RALDH-2 gene is also expressed in specific regions of the developing head, including the tooth buds, inner ear, meninges and pituitary gland, and in several viscera. Administration of a teratogenic dose of RA at embryonic day 8.5 results in downregulation of RALDH-2 transcript levels in caudal regions of the embryo, and may reflect a mechanism of negative feedback regulation of RA synthesis.

Light-mediated retinoic acid production.

Retinoids serve two main functions in biology: retinaldehyde forms the chromophore bound to opsins, and retinoic acid (RA) is the activating ligand of transcription factors. These two functions are linked in the vertebrate eye: we describe here that illumination of the retina results in an increase in RA synthesis, as detected with a RA bioassay and by HPLC. The synthesis is mediated by retinaldehyde dehydrogenases which convert some of the chromophore all-trans retinaldehyde, released from bleached rhodopsin, into RA. As the eye contains high levels of retinaldehyde dehydrogenases, and as the oxidation of retinaldehyde is an irreversible reaction, RA production has to be considered an unavoidable by-product of light. Through RA synthesis, light can thus directly influence gene transcription in the eye, which provides a plausible mechanism for light effects that cannot be explained by electric activity. Whereas the function of retinaldehyde as chromophore is conserved from bacteria to mammals, RA-mediated transcription is fully evolved only in vertebrates. Invertebrates differ from vertebrates in the mechanism of chromophore regeneration: while in the invertebrate visual cycle the chromophore remains bound, it is released as free all-trans retinaldehyde from illuminated vertebrate rhodopsin. RA synthesis occurring as corollary of dark regeneration in the vertebrate visual cycle may have given rise to the expansion of RA-mediated transcriptional regulation.