In vertebrates, symmetric versus asymmetric cleavage of ␤-carotene in the biosynthesis of vitamin A and its derivatives has been controversially discussed. Recently we have been able to identify a cDNA encoding a metazoan ␤,␤-carotene-15,15-dioxygenase from the fruit fly Drosophila melanogaster. This enzyme catalyzes the key step in vitamin A biosynthesis, symmetrically cleaving ␤-carotene to give two molecules of retinal. Mutations in the corresponding gene are known to lead to a blind, vitamin A-deficient phenotype. Orthologs of this enzyme have very recently been found also in vertebrates and molecularly characterized. Here we report the identification of a cDNA from mouse encoding a second type of carotene dioxygenase catalyzing exclusively the asymmetric oxidative cleavage of ␤-carotene at the 9,10 double bond of ␤-carotene and resulting in the formation of ␤-apo-10-carotenal and ␤-ionone, a substance known as a floral scent from roses, for example. Besides ␤-carotene, lycopene is also oxidatively cleaved by the enzyme. The deduced amino acid sequence shares significant sequence identity with the ␤,␤-carotene-15,15-dioxygenases, and the two enzyme types have several conserved motifs. To establish its occurrence in different vertebrates, we then attempted and succeeded in cloning cDNAs encoding this new type of carotene dioxygenase from human and zebrafish as well. As regards their possible role, the apocarotenals formed by this enzyme may be the precursors for the biosynthesis of retinoic acid or exert unknown physiological effects. Thus, in contrast to Drosophila, in vertebrates both symmetric and asymmetric cleavage pathways exist for carotenes, revealing a greater complexity of carotene metabolism.

The egg yolk of vertebrates contains carotenoids, which account for its characteristic yellow color in some species. Such plant-derived compounds, e.g. β-carotene, serve as the natural precursors (provitamins) of vitamin A, which is indispensable for chordate development. As egg yolk also contains stored vitamin A, carotenoids have so far been solely discussed as pigments for the coloration of the offspring. Based on our recent molecular identification of the enzyme catalyzing provitamin A conversion to vitamin A, we address a possible role of provitamin A during zebrafish (Danio rerio) development. We cloned the zebrafish gene encoding the vitamin A-forming enzyme, a β,β-carotene-15,15′-oxygenase. Analysis of its mRNA expression revealed that it is under complex spatial and temporal control during development. Targeted

Forward genetic screens have been instrumental in defining molecular components of visual function. The zebrafish mutant fading vision (fdv) has been identified in such a screen due to defects in vision accompanied by hypopigmentation in the retinal pigment epithelium (RPE) and body melanocytes. The RPE forms the outer most layer of the retina, and its function is essential for vision. In fdv mutant larvae, the outer segments of photoreceptors are strongly reduced in length or absent due to defects in RPE cells. Ultrastructural analysis of RPE cells reveals dramatic cellular changes such as an absence of microvilli and vesicular inclusions. The retinoid profile is altered as judged by biochemical analysis, arguing for a partial block in visual pigment regeneration. Surprisingly, homozygous fdv vision mutants survive to adulthood and show, despite a persistence of the hypopigmentation, a partial recovery of retinal morphology. By positional cloning and subsequent morpholino knock-down, we identified a mutation in the silver gene as the molecular defect underlying the fdv phenotype. The Silver protein is required for intralumenal fibril formation in melanosomes by amylogenic cleavage. Our data reveal an unexpected link between melanosome biogenesis and the visual system, undetectable in cell culture.

An enzyme-based cyclic pathway for trans to cis isomerization of the chromophore of visual pigments (11-cis-retinal) is intrinsic to vertebrate cone and rod vision. This process, called the visual cycle, is mostly characterized in rod-dominated retinas and essentially depends on RPE65, an alltrans to 11-cis-retinoid isomerase. Here we analysed the role of RPE65 in zebrafish, a species with a cone-dominated retina. We cloned zebrafish RPE65 and showed that its expression coincided with photoreceptor development. Targeted gene knockdown of RPE65 resulted in morphologically altered rod outer segments and overall reduced 11-cis-retinal levels. Cone vision of RPE65-deficient larvae remained functional as demonstrated by behavioural tests and by metabolite profiling for retinoids. Furthermore, all-trans retinylamine, a potent inhibitor of the rod visual cycle, reduced 11-cis-retinal levels of control larvae to a similar extent but showed no additive effects in RPE65-deficient larvae. Thus, our study of zebrafish provides in vivo evidence for the existence of an RPE65-independent pathway for the regeneration of 11-cis-retinal for cone vision.

Vitamin A derivatives (retinoids) are essential components in vision; they contribute to pattern formation during development and exert multiple effects on cell differentiation with important clinical implications. All naturally occurring vitamin A derives by enzymatic oxidative cleavage from carotenoids with pro-vitamin A activity. To become biologically active, these plant-derived compounds must first be absorbed, then delivered to the site of action in the body, and metabolically converted to the real vitamin. Recently, molecular players of this pathway were identified by the analysis of blind Drosophila mutants. Similar genome sequences were found in vertebrates. Subsequently, these homologous genes were cloned and their gene products were functionally characterized. This review will summarize the advanced state of knowledge about the vitamin A biosynthetic pathway and will discuss biochemical, physiological, developmental and medical aspects of carotenoids and their numerous derivatives.

For vertebrate development, vitamin A (all-trans retinol) is required in quantitative different amounts and spatiotemporal distribution for the production of retinoic acid, a nuclear hormone receptor ligand, and 11-cis retinal, the chromophore of visual pigments. We show here for zebrafish that embryonic retinoid homeostasis essentially depends on the activity of a lecithin:retinol acyltransferase (Lratb). During embryogenesis, lratb is expressed in mostly non-overlapping domains opposite to retinal dehydrogenase 2 (raldh2), the key enzyme for retinoic acid synthesis. Blocking retinyl ester formation by a targeted knock down of Lratb results in significantly increased retinoic acid levels, which lead to severe embryonic patterning defects. Thus, we provide evidence that a balanced competition between Lratb and Raldh2 for yolk vitamin A defines embryonic compartments either for retinyl ester or retinoic acid synthesis. This homeostatic mechanism dynamically adjusts embryonic retinoic acid levels for gene regulation, concomitantly sequestering excess yolk vitamin A in the form of retinyl esters for the establishment of larval vision later during development.The biological importance of retinoids (vitamin A or alltrans retinol (ROL) 4 and its derivatives) for vertebrate development has long been known, because both deprivation and exposure to excess retinoids cause major embryonic abnormalities. The ROL derivative all-trans retinoic acid (RA) is a hormonal signaling molecule that acts in developing and adult tissues (1). RA regulates gene expression via heterodimeric nuclear receptors, the RA receptors and the retinoid X receptors (2, 3). Both are ligand-dependent transcription factors belonging to the superfamily of nuclear hormone receptors (4). RA is generated from maternal ROL via all-trans retinal (all-trans RAL) by a stepwise enzymatic oxidation. Hereby, the second oxidation step catalyzed by retinal dehydrogenases (Raldhs) is thought to be rate-limiting (5, 6). The first step in the turn-off mechanism of retinoid signaling is the catabolism of RA by a class of cytochrome P450 enzymes (Cyp26s) to more polar products, such as 4-hydroxy RA or 4-oxo RA (7).The catabolic control of RA levels via Cyp26s is not sufficient to explain all aspects of retinoid homeostasis, considering for example the key role of vitamin A in vision. In photoreceptors, ROL is required for the synthesis of 11-cis retinal (11-cis RAL), the chromophore of visual pigments (8), in much higher amounts than for RA synthesis in gene regulation. This quantitatively different requirement for ROL calls for a mechanism that maintains the homeostasis between RA and 11-cis RAL synthesis. To elucidate the underlying control mechanism of retinoid homeostasis, we used the zebrafish as a model. In this oviparous vertebrate, yolk retinoids must be proportioned during embryonic development to adequately support both these processes. We found that sequestration of retinyl esters (RE) is essential for retinoid homeostasis during embryonic development. We mol...

The retinoic acid molecule, a vitamin A derivative, is of key importance for eye and photoreceptor development in vertebrates. Several studies have provided evidence that the ventral part of the retina is particularly susceptible to impairment in retinoid signalling during the period of its development. In zebrafish, targeted gene knockdown of beta,beta-carotene-15,15'-oxygenase (bcox), the key enzyme for vitamin A formation, provokes a loss of retinoid signalling during early eye development that results in microphthalmia at larval stages. Using this model, we analysed the consequences of this for the retinal morphology of the fish larvae in structural details. Our analyses revealed that rods and cones do not express photoreceptor specific proteins (rhodopsin, peanut agglutinin, zpr1) in the peripheral retina. The photoreceptors in the central retina showed shortened outer segments, and electron dense debris in their intermembranal space. The number of phagosomes was increased, and cell death was frequently observed in the outer nuclear layer. Furthermore, the number of Muller cells was significantly reduced in the inner nuclear layer. Thus, we found that the lack of retinoid signalling strongly effects photoreceptor development in the ventral and dorsal retina. In addition, shortened outer segments and cell death of the remaining photoreceptors in the central retina indicate that there is an ongoing need for retinoid signalling for photoreceptor integrity and survival at later developmental stages.

Partially purified acetyl-CoA carboxylase was covalently bound to a Sepharose 4B matrix. Although aggregation was thus prevented, the enzymic activity was stimulated by citrate and isocitrate.