Peter McCaffery

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.

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 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.

Influence of the choroid plexus on cerebellar development: analysis of retinoic acid synthesis

The choroid plexus of the fourth ventricle is conspicuous both in location and size: it protrudes over the outer hindbrain, closely apposed to the caudal external surface of the cerebellum, and it is disproportionately large early on. While the developing cerebellum is known to respond to retinoic acid (RA), it does not express significant levels of RA synthesizing enzyme. Retinaldehyde dehydrogenase levels in the choroid plexus, however, are very high, with maxima during the pre- and postnatal periods of cerebellar morphogenesis. Explants assays demonstrate release of a neurite-outgrowth promoting activity from the choroid plexus, whose levels parallel the levels of RA synthesizing enzyme here, and which can be mimicked by RA. These observations characterize the choroid plexus as a paracrine, growth-promoting organ for the developing cerebellum, with the effects mediated through temporally regulated RA production.

Retinaldehyde dehydrogenase 2 and Hoxc8 are required in the murine brachial spinal cord for the specification of Lim1+ motoneurons and the correct distribution of Islet1+ motoneurons

Retinoic acid (RA) activity plays sequential roles during the development of the ventral spinal cord. Here, we have investigated the functions of local RA synthesis in the process of motoneuron specification and early differentiation using a conditional knockout strategy that ablates the function of the retinaldehyde dehydrogenase 2 (Raldh2) synthesizing enzyme essentially in brachial motoneurons, and later in mesenchymal cells at the base of the forelimb. Mutant (Raldh2L–/–) embryos display an early embryonic loss of a subset of Lim1+ brachial motoneurons, a mispositioning of Islet1+ neurons and inappropriate axonal projections of one of the nerves innervating extensor limb muscles, which lead to an adult forepaw neuromuscular defect. The molecular basis of the Raldh2L–/– phenotype relies in part on the deregulation of Hoxc8, which in turn regulates the RA receptor RARβ. We further show that Hoxc8 mutant mice, which exhibit a similar congenital forepaw defect, display at embryonic stages molecular defects that phenocopy the Raldh2L–/– motoneuron abnormalities. Thus, interdependent RA signaling and Hox gene functions are required for the specification of brachial motoneurons in the mouse.

Retinoic Acid Synthesizing Enzymes in the Embryonic and Adult Vertebrate

The oxidation of retinaldehyde to retinoic acid (RA) provides the retinoid form of highest potency for a variety of cellular systems. RA has been implicated in many processes, such as growth and differentiation of epithelia in the adult organism (De Luca 1991), and determination of the antero-posterior axis for the limb bud (Eichele and Thaller 1987; Tickle et al. 1982) and the entire body of the vertebrate embryo (Durston et al. 1989; Hogan, Thaller, and Eichele 1992). In addition, RA is thought to promote neuronal survival, differentiation and neurite outgrowth (Haskell et al. 1987; Quinn and De Boni 1991; Wuarin, Sidell, and De Vellis 1990). RA exerts its effects by binding to specific nuclear receptors that regulate transcription. The diversity in RA actions is commonly attributed to differences in local expression patterns of different receptors and cytoplasmic binding proteins that modify the availability of intracellular RA (Giguere 1994). In addition, however, retinoid metabolism may contribute significantly to local diversity in RA actions. Retinoid metabolism includes the processes of precursor circulation and cellular uptake mediated by binding proteins, the reversible oxidation of retinol to retinaldehyde, the irreversible oxidation of retinaldehyde to RA, and RA degradation. Here we focus on the enzymes that mediate the oxidation of retinaldehyde to RA.

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.

Retinoic acid and development of the retina

Abstract Retinoic acid (RA) is known as the activating trigger for a large number of processes in developing and mature vertebrates, and it plays a pivotal role in eye development. We present here a brief review of the RA system in general, and we summarize the evidence for a determining role of RA in the embryonic eye. The earliest and most significant ocular feature influenced by RA is the dorso-ventral axis. A lasting differential expression of different RA generating enzymes along the retinal dorso-ventral axis then creates very high endogenous RA levels, as well as a ventro-dorsal RA gradient, features that are likely to direct morphogenesis along this axis in the embryonic eye. RA is also likely to play a significant role in the function of the mature eye, as some of the chromophore released from photo-bleached rhodopsin is converted to RA, a mechanism for light to directly influence gene expression. The pivotal role of RA in eye morphogenesis may represent a developmental correlate of an evolutionary origin of RA-mediated transcriptional regulation from retinoid usage in vision.

Sources and sink of retinoic acid in the embryonic chick retina: distribution of aldehyde dehydrogenase activities, CRABP-I, and sites of retinoic acid inactivation

Previous experiments in mice and zebrafish led to the hypothesis that an asymmetric distribution of the transcriptional activator retinoic acid (RA) causes ventral-dorsal polarity in the vertebrate eye anlage. A high concentration of RA in the ventral retinal neuroepithelium has been suggested to induce developmental events that finally establish topographic order in the retinotectal projection along the vertical eye axis. In the present study we have investigated potential sources and sinks of RA during embryonic development of the chick retina. At embryonic day (E)1 to E2, when the spatial determination of the eye primordia takes place, no RA synthesis by aldehyde dehydrogenases was detectable, and neither immunoreactivity for retinaldehyde dehydrogenase RALDH-2 nor for cellular retinoic acid binding protein CRABP-I was observed. These components of RA signal transduction appeared in the eye between E3 and E5. At later stages, RA-measurements with a reporter cell line showed highest synthesis in the retinal pigment epithelium (RPE) and at the ventral and dorsal poles of the retina. RA degradation occurred mostly in a horizontal region in the middle of the retina with only small differences along the nasal-temporal axis. CRABP-I immunoreactivity appeared first in differentiating retinal ganglion cells with no indication of a spatial gradient across the ventral-dorsal eye axis. RA-production depended on three NAD+-dependent enzyme activities, which could be competitively inhibited by citral. One enzyme, located in the dorsal retina (corresponding to mouse RALDH-1), and one enzyme in the RPE (RALDH-2) were aldehyde dehydrogenases of the same molecular weight (monomers about 55 kDa) but with different isoelectric points (6.5-6.9; 4.9-5.4). The third RA-synthesizing activity (pI 6.0-6.3) was limited to the ventral retina, and likely corresponded to mouse RALDH-3. The restricted localization of retinoid-metabolizing activities along the dorsal-ventral axis of the embryonic chick retina does support the idea that RA is involved in dorsal-ventral eye patterning. However, the late time of appearance of aldehyde dehydrogenase activities and CRABP-I points to functions in cellular differentiation that are distinct from the initiation of the dorsal-ventral polarity.

Retinoic acid synthesis in the developing retina

Neuronal information in the vertebrate brain is represented in the form of topographical maps: there are multiple topographical representations in the brain of the visual world, of auditory space, of the tactile body surface and of movement directions. Information processing in the framework of maps is a general feature of the nervous system throughout vertebrates and even in lower species. The main advance through evolution is not in the principle of mapping, but in the number and specialization of maps. The maps are formed in the embryonic brain due to a capacity of neuronal processes from one brain area to search out precisely their address in the next area. The molecular basis of this neuronal specificity is still largely unknown. The main working hypothesis, formulated for the example of the visual system, postulates two sets of cell-surface markers, one graded in the antero-posterior, the other in the dorso-ventral axis of the retinal maps, which enable optic growth cones to match up with corresponding markers in their targets (Sperry, 1963). The expression of such graded markers needs to be preceded by an asymmetry in gene transcription. The enzymes described here provide a direct access to this early step of neuronal specification in the retina, as their product, retinoic acid, regulates gene transcription through nuclear receptors (Chambon et al., 1991).