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内容記述 |
To understand the molecular basis for the establishment of regional specificity during retinal development, it is important to make up a catalogue of the molecules with asymmetrical expressions in the developing retina. To address this issue, a large-scale screening using Restriction Landmark cDNA Scanning (RLCS) was performed along the antero-posterior or dorso-ventral axis in the chick retina. A number of molecules, which showed asymmetrical expression along the respective axes in the developing retina, were isolated. Among them, I identified two aldehyde dehydrogenases; one was a ventral retina-specific novel clone V/Bgl II#1, and the other was already known dorsal retina specific retinaldehyde dehydrogenase 1 (RALDH-1). This dissertation mainly describes the identification and characterization of the V/Bgl II#1.<br /> In the developing retina, a retinoic acid (RA) gradient (ventral high-dorsal low) along the dorso-ventral axis is considered to be important for the regional specification, especially for the ventral retinal formation. For example, deficiency of vitamin A in rat embryos results in abnormalities in eye formation: the eyes are small or missing, and mainly a reduction in size of the ventral half is observed. Symptoms similar to vitamin A deficiency have been observed in double knock-out mice for RA receptors, which the ventral half of the retina is reduced in size. The RA gradient has been thought to result from the asymmetrical distribution of retinaldehyde dehydrogenases: one is RALDH-1, and the other is a ventral retina-specific enzyme, which has not been cloned. First, I addressed whether V/Bgl II#1 is the ventral RA-generating enzyme. Using a retinoid-responsive reporter cell line, I confirmed that V/Bgl II#1 has the activity to produce RA from retinaldehyde. I thus concluded this molecule as the ventral-specific RALDH, and named it retinaldehyde dehydrogenase 3 (RALDH-3). A search for similar sequences in DNA databases showed that RALDH-3 has the highest similarity (86% identical) with human aldehyde dehydrogenase 6 (hALDH6). The mouse homologue of V/Bgl II#1 was then isolated from a neonatal mouse eye cDNA library. Chick and mouse RALDH-3, and hALDH6 all consist of 512 amino acid residues and showed high similarity with each other: the mouse RALDH-3 is 85%, 94% identical to the chick and hALDH6, respectively. The mouse Raldh-3 expression was detected by RT-PCR in some adult mouse tissues including kidney, stomach and salivary gland, which are known to express hAldh6 at high levels. This suggests that RALDH-3 is the orthologue of hALDH6. <br /> Next, I performed in situ hybridizations to gain insight into the spatio-temporal expression of Raldh-3 and Raldh-1 during chick and mouse embryonic eye development. Raldh-3 was expressed first in the surface ectoderm overlying the ventral portion of the prospective eye region, and then in the ventral retina. In mouse, Raldh-3 expression was also observed in the invaginating lens pit. On the other hand, the Raldh-1 expression began in the dorsal retina nearly at the same time with Raldh-3 expression in the ventral retina. The Raldh-1 expression in chick was restricted to the dorsal retina throughout the development, while in mouse it was also observed in the ventral retina and lens vesicle at the later developmental stage. The Raldh-3 expression in the lens pit seemed to be replaced by the Raldh-1 in the lens vesicle. The occurrence of two RALDH genes that are controlled differently may thus underlie the specific development of the dorsal and ventral retina.<br /> The paired-like homeobox-containing transcription factor Pax6 is known to be a master gene of the eye development. Recently, it was reported for Pax6 mutant mice that the local production of RA in the eye region and nasal region is disrupted. This suggests that Pax6 regulates retinoid formation in the eye region as well as in the nasal region. I therefore examined the expressions of Raldh-1 and Raldh-3 in Pax6 mutant rat (rSey2) embryos. I found that E11.5 Pax6 mutants are devoid of Raldh-3 expression. At E13.5 no Raldh-1 expression was detected in the mutant, though strong Raldh-1 expression was observed in the dorsal retina in the wild type.<br /> In summary, I identified a novel RALDH, RALDH-3, which is responsible for RA production in the ventral eye region. The results obtained in this study indicated that: 1) Raldh-3 expression in the surface ectoderm is responsible for the RA-gradient formation along the dorso-ventral axis during the early eye development, 2) Raldh-1 and Raldh-3 are individually involved in the dorsal and ventral retinal development, respectively, 3) mouse Raldh-l and Raldh-3 might have some additional roles in the eye development as compared with chick orthologues, and 4) Raldh-3 is a downstream target of Pax6, and Pax6 exerts its functions at least in part through R7X produced by RALDH-3. |
要旨・審査要旨 / Abstract, Screening Result (275.8 kB)
