Human Molecular Genetics, 2000, Vol. 9, No. 19 2781-2788
© 2000 Oxford University Press
Oa1 knock-out: new insights on the pathogenesis of ocular albinism type 1
1Telethon Institute of Genetics and Medicine (TIGEM), San Raffaele Biomedical Science Park, Via Olgettina 58, I-20132 Milan, Italy, 2Department of Experimental Medicine, Anatomy Section, University of Genova, I-16132 Genova, Italy, 3University College London, Institute of Ophthalmology, London, UK, 4Retinal Electrodiagnostics Research Group, University Eye Hospital, D-72076 Tübingen, Germany, 5St George’s Hospital Medical School, Cranmer Terrace, London SW17 0RE, UK and 6Università Vita e Salute, San Raffaele, I-20132 Milan, Italy
Received 3 July 2000; Revised and Accepted 17 September 2000.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Ocular albinism type I (OA1) is an X-linked disorder characterized by severe reduction of visual acuity, strabismus, photophobia and nystagmus. Ophthalmologic examination reveals hypopigmentation of the retina, foveal hypoplasia and iris translucency. Microscopic examination of both retinal pigment epithelium (RPE) and skin melanocytes shows the presence of large pigment granules called giant melanosomes or macromelanosomes. In this study, we have generated and characterized Oa1-deficient mice by gene targeting (KO). The KO males are viable, fertile and phenotypically indistinguishable from the wild-type littermates. Ophthalmologic examination shows hypopigmentation of the ocular fundus in mutant animals compared with wild-type. Analysis of the retinofugal pathway reveals a reduction in the size of the uncrossed pathway, demonstrating a misrouting of the optic fibres at the chiasm, as observed in OA1 patients. Microscopic examination of the RPE shows the presence of giant melanosomes comparable with those described in OA1 patients. Ultrastructural analysis of the RPE cells, suggests that the giant melanosomes may form by abnormal growth of single melanosomes, rather than the fusion of several, shedding light on the pathogenesis of ocular albinism.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Albinism is a generic designation for a variety of clinical syndromes exhibiting hypomelanosis. It includes several heritable metabolic defects in the pigment cell (melanocyte) system of the eye and integument. Two general forms of albinism are recognized: oculocutaneous albinism (OCA) and ocular albinism (OA). In OCA, the eye, skin and hair manifest phenotypic abnormalities, whereas OA affects primarily the eye. The classification of albinism includes at least 10 types of OCA and four types of OA. Most of these result from the involvement of different genes, although some may be due to allelic mutations at the same locus (1).
Ocular albinism of the Nettleship–Falls type (OA1; MIM 300500) is the most common form of ocular albinism (2). This disorder is transmitted as an X-linked recessive trait, with affected males showing the complete phenotype and heterozygous females showing only minor signs of disease (3). Affected males with OA1 have an extremely severe reduction of visual acuity, which represents a major handicap. They also manifest horizontal and rotary nystagmus, strabismus and marked photophobia. Ophthalmologic examination reveals foveal hypoplasia, hypopigmentation of the retina and iris translucency (1,4,5). As indicated by the non-symmetric pattern of the visual evoked potential, these patients have misrouting of the optic fibres at the chiasm, resulting in a loss of stereoscopic vision (6,7). Most female carriers for OA1 can be clinically identified by a typical mud splattered mosaic pattern of depigmentation in the periphery of the fundus (3), suggesting that the OA1 gene is subject to X-inactivation.
Cutaneous changes are minimal in OA1 and mild hypopigmentation of the skin is found in rare cases. Microscopic examination of melanocytes, in both retinal pigment epithelium (RPE) and in the skin, reveals the presence of large pigment granules called giant melanosomes or macromelanosomes (4,8–10). This finding helps distinguish between OA1 and other types of albinism and suggests that the underlying defect in OA1 is an abnormality in melanosome formation.
The locus for OA1 has been assigned to the Xp22.3 region through both linkage (11) and deletion mapping (12,13). Using a positional cloning approach, the gene responsible for OA1 has been isolated (14). The OA1 transcript measures 
1.6 kb and was readily detected by northern blot analysis, exclusively in RNA preparations from pigment cells, such as melanocytes, melanoma and RPE. The OA1 predicted protein product is 404 amino acids long and displays several putative transmembrane domains (14). The OA1 protein product was detected in human pigment cells as a melanosomal membrane glycoprotein (15). These observations suggest that OA1 may be involved in melanosome organelle formation. Recent data from our laboratory demonstrated that OA1 displays structural homology with G protein-coupled, seven transmembrane domain receptors (16). Co-immunoprecipitation of OA1 with Gß and G
i in melanocyte extracts demonstrated its specific interaction with heterotrimeric Gi proteins. Furthermore, at variance with all the other known members of this family of receptors, OA1 is exclusively localized in intracellular organelles. Thus, the putative OA1 receptor may represent a ‘sensor’ of a yet-unidentified intra-melanosomal ligand, regulating organelle biogenesis and maturation through activation of heterotrimeric G proteins on the cytoplasmic side of the melanosomal membrane.
To investigate the biological function of OA1 we have cloned the mouse orthologue of OA1 (17) and generated a mouse in which the gene encoding the Oa1 protein was inactivated. Analysis of Oa1-deficient mice revealed hypopigmentation of the fundus, presence of macromelanosomes in the RPE and misrouting of the optic fibres at the chiasm. Oa1-deficient mice thus appear to be a suitable transgenic animal model of ocular albinism type 1 and will allow further unravelling of the pathogenic mechanisms underlying this disease.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Generation and macroscopic analysis
A targeting vector was constructed in which the first exon of the Oa1 gene was replaced by an Hprt cassette (Fig. 1) (a kind gift of Dr M.M. Matzuk, BCM, Houston, TX). The first exon was deleted because several OA1 patients carry missense mutations as well as insertions and deletions in this exon; this deletion also eliminates the start codon. After electroporation into AB2.2 embryonic stem (ES) cells (a kind gift of Dr A. Bradley, BCM, Houston, TX) seven recombinant ES clones (4.6%) were identified as positive for gene disruption based on the predicted size of the targeted allele (Fig. 1). We obtained 13 chimeric mice from two ES clones (1.61 and 1.77) that were crossed with C57BL/6 mice to produce heterozygous carrier females. Four carrier females were generated and mated to either 129/Sv or C57BL/6 males to generate Oa1–/Y hemizygous male mice. The genetic background did not significantly influence the phenotype described below.
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A normal progeny sex ratio was observed, indicating that Oa1–/Y hemizygotes are not embryonic lethal. Northern blot analysis of RNA from primary melanocyte cultures demonstrated the absence of the Oa1 transcript in Oa1 mutant mice, whereas the transcript was detectable in wild-type and heterozygous mice (data not shown). On macroscopic examination, Oa1–/Y mice were anatomically normal and indistinguishable by coat colour from their littermates. We observed no evident differences between Oa1+/Y and Oa1–/Y mice in survival, fertility, gross physical appearance and organ morphology. Ophthalmologic examination revealed hypopigmentation of the fundus, resulting in a more pronounced pattern of choroidal vasculature (data not shown).
Histological examination
Histological analysis of retina, brain, heart, liver, kidney, adrenal gland and lung from wild-type, heterozygous and mutant adult animals, revealed no obvious anomalies.
Given that RPE is the structure primarily affected in OA1 patients, we focused our analysis on the eyes of embryos and postnatal animals collected from stages at embryonic day (E) 11.5, E12.5, E15.5, E16.5, E17.5, post-partum day (P) 1, P5, P7 and adult. We selected these specific stages based on the timing of RPE differentiation and development of macromelanosomes. Pigment first appears in the retinal region around E11–E11.5 of gestation (18,19). Analysis of the eyes showed the absence of giant melanosomes in the RPE of mutant mice from E11.5 to 17.5. In mutant mice, unlike normal littermates, abnormally large melanin granules, as well as normal sized melanosomes, were dispersed within the pigment epithelium of the iris, ciliary body and retina at P1 (the first 24 h following birth). At P7 no abnormalities were found in the ocular features of control littermate mice, whereas the majority of melanosomes in the RPE of Oa1–/Y mice displayed a giant phenotype. The total number of mature fully pigmented melanosomes per cell profile at stages E16.5 and P7 was comparable to normal, as revealed by computer-assisted counting of pigmented organelles (IPlab image analysis software) (Table 1). Examination of the eyes of Oa1–/Y adult animal, showed the RPE to be more lightly pigmented than in controls. The RPE analysis demonstrated the presence of giant pigment granules comparable to those described in OA1 patients (Fig. 2) (4). No individual macromelanosomes were observed in adult skin, hair and uveal melanocytes.
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Ultrastructural analysis
Ultrastructural analysis of the RPE in Oa1–/Y mice showed normal sized melanosomes at stages from E12.5 to P1 (Fig. 3). Macromelanosomes were first detected at P1 and their number increased progressively until adulthood (Fig. 3). At P7 the majority of the melanosomes displayed a giant phenotype. The average diameter of macromelanosomes was about three times the diameter of normal melanosomes observed in wild-type animals (18.8 versus 4.6 µm). These data confirmed that the subcellular defects observed in the RPE of Oa1–/Y mice are consistent with those found in the human disease (4).
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No evidence of intermediate stages of melanosome–melanosome fusion was found. Macromelanosomes in the RPE of Oa1–/Y mice showed several paler round areas embedded within the electron-dense melanin, similarly to human macromelanosomes (4). Within the electron-dense melanin, we also identified a central core region which closely resembled the structure of a normal membrane-free melanosome (i.e. elongated matrix fibres coated by pigment) (Fig. 4).
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Together, the presence of a single core melanosome within the giant pigmented organelles, the normal number of pigmented melanosomes within the cell and the absence of intermediates of melanosome fusion suggest that giant melanosomes derive from abnormal growth of single organelles, rather than from the fusion of normal melanosomes.
Electrophysiological analysis
To analyse potential retinal impairment, Ganzfeld electroretinograms (ERGs) were recorded at increasing stimulus intensities under scotopic (dark-adapted) and photopic (light-adapted) conditions. Due to characteristic contributions of photoreceptors, bipolar cells and amacrines, the ERG is a good indicator of retinal function below the ganglion cell level. In Oa1–/Y mice, the ERGs did not reveal any differences from control animals over the entire intensity range (Fig. 5). This result indicates that retinal function is not affected by Oa1 disruption.
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The optic chiasm
To investigate whether the Oa1–/Y mice show a misrouting of the optic fibres similar to that observed in OA1 patients, we have examined the chiasmatic pathways in mutant and control animals. A single unilateral intraocular injection of horseradish peroxidase (HRP) was made into the left eye of each mouse. In all the mice used in this study the major component of the uncrossed retinal projection terminated in the lateral geniculate nucleus (LGN) and in the superior colliculus (SC). In the Oa1+/Y animals there was a clear region of terminal label in the LGN ipsilateral to the injected eye that extended in an orderly manner along the rostrocaudal length of the nucleus. Terminal label on this side of the brain was also identified in the pretectal region and in the rostral border of the SC. There was considerably more label in the visual pathway contralateral to the injected eye than ipsilateral to it. In the crossed pathway, label filled the majority of the LGN, with the exception of a region roughly corresponding to the area of label in the LGN ipsilateral to the injected eye (Fig. 6). In the Oa1 mutant mice the size of the uncrossed pathway was reduced in favour of the crossed pathway. In the visual pathway ipsilateral to the injected eye label was very sparse. The location of the label in the LGN was similar to that in the wild-type animals, but reduced in density. In the visual pathway contralateral to the injected eye, label was continuous across the LGN, with only an occasional thinning in regions corresponding to the location of the uncrossed projection on the other side of the brain. In detail, the Oa1+/Y animals have a mean ipsilateral volume of terminal label in the LGN of 0.3 mm3 whereas the Oa1–/Y mice have a mean of 0.019 mm3; this difference is statistically significant at a 5% level (Mann–Whitney U-test). The changes in the laterality of the configuration of the retinofugal pathway were independent of the volume of the LGN, as the size of this nucleus was similar in both mutant and wild-type animals.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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OA1 is the commonest form of ocular albinism. It results in severe visual handicap in affected patients. One characteristic feature of OA1 patients is the presence of giant melanosomes (macromelanosomes) in melanocytes from RPE and skin (4,8–10). Recent data from our laboratory suggest that OA1 may function as a G protein-coupled receptor, the first to be exclusively localized in intracellular organelles (16). Although the precise role of the Oa1 protein remains illusive, it is clear that the absence of the Oa1 gene product has serious implications for melanosome number, structure and melanization, supporting the hypothesis that it plays a central role in melanosome biogenesis. To unravel its function, we have generated a mouse model of the disease. A null mutant was obtained by deletion of the first exon in the Oa1 gene. The knock-out males (Oa1–/Y) were viable and fertile, with coat and eye colour indistinguishable from those of wild-type littermates. As described in humans affected by OA1, the fundus was hypopigmented and in the RPE giant melanosomes were present. Furthermore, the retinofugal pathway displayed a misrouting of optic fibres.
Electroretinographic analysis of retinal function did not show any abnormalities in the Oa1–/Y mice. This is similar to the human phenotype, where the ERG is normal (this can in some cases even be elevated owing to additional trans-scleral illumination) (20–22). As mice do not have a central retinal specialization, it was not possible to study this aspect of the disease.
The availability of a mouse model for OA1 is an important tool in the ultrastructural analysis of the RPE throughout development, enabling an analysis of the pathogenesis of this disease and macromelanosome biogenesis. Analysis of RPE pathology during human development is clearly difficult. However, a single report describing RPE histology, in a male fetus of 21 weeks’ gestation affected by OA1, demonstrated the presence of both normal sized and giant pigment granules in the RPE, in most cases one giant granule per individual RPE cell was observed (9).
We performed the analysis at stages from E11.5 to adult. Ultrastructural analysis of the eyes showed an absence of giant melanosomes in the RPE from E11.5 to E17.5. At these stages melanosome number, structure and degree of maturation were similar in both wild-type and mutant animals. Macromelanosomes were first detected at P1 and their number increased progressively until adulthood, when the majority of the melanosomes displayed a giant phenotype. No macromelanosomes have been detected in hair or skin melanocytes. We believe that, as in these cells the mature melanosomes are transferred from melanocytes to keratinocytes, they may not remain in melanocytes long enough to display the mutation effect.
The currently accepted model for melanosome biogenesis invokes a bipartite pathway (23), in which tyrosinase-charged vesicles, budding off the trans-Golgi network, fuse with smooth endoplasmic reticulum-derived premelanosomes (stage I–II melanosomes), initiating the process of melanization. Macromelanosomes were formed due to the failure of premelanosomes to separate from the endoplasmic reticulum–Golgi system continuum, with enzymes and structural proteins steadily accumulating and causing progressive organelle distension (24). However, recent data (25) suggest a different pathway in melanosome genesis, including endosomes as intermediate steps of protein sorting to the pigmented organelles. Our data do not support the hypothesis that macromelanosomes are derived by endoplasmic reticulum enlargement. They strongly suggest that normal and giant melanosomes undergo a similar formation and maturation process, excluding the possibility that fusion of two or more melanosomes could be involved. In particular, we have found: (i) the presence of a single core melanosome within the giant pigmented organelles; (ii) that the total number of mature pigmented melanosomes per cell is unchanged until P7 and slightly reduced in adult samples; and (iii) a lack of intermediates of melanosome–melanosome fusion. Based on these findings, the role of Oa1 in normal melanogenesis could be in the final stages of organelle growth and maturation. Indeed, giant melanosomes were detected in mutant animals only after birth when it is generally accepted that melanogenesis in the RPE is completed (26–28). It is not clear at what level of melanosome maturation the Oa1 inhibitory activity might be exerted. As the putative ligand-binding site is orientated towards the melanosome lumen, one could envisage a role as a sensor of melanin synthesis. However, as a giant organelle requires extra membrane, an alternative hypothesis could be Oa1 regulates membrane traffic towards the melanosome.
To investigate whether the Oa1–/Y mice display misrouting of optic fibres at the chiasm similarly to OA1 patients, we examined chiasmatic pathways in both mutant and control animals. In hypopigmented mammals, many developing fibres from the temporal retina are inappropriately routed at the chiasm into the contralateral hemisphere, where they form abnormal binocular maps in the LGN (29). In rodents, cells projecting to each hemisphere are mixed in the temporal retina. In albino rodents there is a reduction in the number of ipsilaterally projecting cells in favour of those projecting contralaterally (30). Analysis of Oa1–/Y mice showed a reduction in the size of the uncrossed pathway, consistent with there being fibre misrouting. The absence of macromelanosomes when the first retinal axons enter the chiasmic region (E12.5) and later, when both crossed and uncrossed axons are present (E16), suggests that there is no direct causative effect of macromelanosomes on the abnormal development of these pathways.
Based on the hypopigmentation of the ocular fundus, the macromelanosomes in the RPE and the inappropriate crossing of the ipsilateral component in the chiasm in adult null mutant animals, we believe that Oa1 mutant mice constitute an authentic counterpart for OA1. Finally, by mating Oa1–/Y mice with other albino mutants, we can obtain mice carrying two homozygous mutations. The generation of double mutants will show whether the Oa1 null phenotype can be modified by the absence of proteins involved in melanin synthesis (e.g. tyrosinase) or melanosome biogenesis. We could, for instance, distinguish between a putative Oa1 inhibitory role in controlling melanin synthesis versus membrane traffic (i.e. evaluate the occurrence of giant melanosome formation in the absence of melanin synthesis), by mating Oa1–/Y with tyrosinase-defective albino mice or with mice carrying mutations affecting membrane traffic, such as mocha. Data collected from studies of double mutants could open new approaches for phenotype rescue in albino patients. These mice may provide a suitable model for the evaluation of a possible in vivo correction of the OA1 defect.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Gene targeting
Three genomic clones encompassing the whole Oa1 coding region were obtained by screening a mouse 129/SvEv phage library (Stratagene, La Jolla, CA) (17) using the complete human OA1 cDNA as a probe. Three-and-a-half kilobases of sequence upstream of exon 1 and 3 kb of sequence downstream of exon 1 were cloned into the PGK-hprt (where PGK is the phosphoglycerate kinase I promoter) expression cassette. An MC1-tk (thymidine kinase) expression cassette was added to the targeting vector to allow negative selection.
Linearization of the vector was performed using an XhoI unique site. Electroporation in AB2.2 ES cells was done as previously described (31). Oa1 mutant cells derived from two homologously recombined clones were injected into C57Bl/6 blastocysts to obtain chimeric mice. Genotypes of ES cells and mouse tails were analysed by Southern blotting after EcoRV or XbaI digestion. Both the 5' and 3' probes were located outside the flanking regions used in the targeting vector and consisted of a 350 bp ApaII–XbaI fragment and a 500 bp BamHI–XbaI fragment, respectively.
ERGs
ERGs were obtained according to previously described procedures (32). In summary, 6- to 7- week-old mice were dark-adapted overnight (at least 6 h) prior to the experiments and their pupils dilated. Anaesthesia was induced by subcutaneous injection of ketamine (66.7 mg/kg), xylazine (11.7 mg/kg) and atropine (1 mg/kg). Silver needle electrodes served as reference (forehead) and ground (tail) and gold wire ring electrodes as active electrodes. The ERG equipment consisted of a Ganzfeld bowl, a DC amplifier and a PC-based control and recording unit (Toennies Multiliner Vision; Jaeger/Toennies, Höchberg, Germany). ERGs were recorded from both eyes simultaneously. Bandpass filter cut-off frequencies were 1 and 300 Hz. Single flash recordings were obtained both under dark-adapted (scotopic) and light-adapted (photopic) conditions. Light adaptation was performed with a background illumination of 30 cd·s/m2 presented for 10 min. Stimuli were presented with increasing intensities, reaching from 10–4 cs/m2 to 25 cd·s/m2, divided into 10 steps of 0.5 and 1 log cd·s/m2. Ten responses were averaged with an inter-stimulus interval (ISI) of 5 or 17 s (for 1, 3, 10, 25 cd·s/m2).
Histological analysis of pheripheral organs
Adult animals were fixed, under pentobarbital anaesthesia, by cardiac perfusion with 4% paraformaldehyde in phosphate-buffered saline (PBS). All major organs were removed and embedded in paraffin for general pathology examination by light microscopy.
Histological and ultrastructural analysis of the eyes
Pregnant animals were killed and fetuses were removed at different gestational stages. For light microscopy, fetuses were killed by decapitation and the head was placed in Davidson solution followed by embedding in paraffin.
For ultrastructural analysis, the eyes were removed and placed in cacodylate buffer containing 2.5% glutaraldehyde, postfixed in osmium tetroxide, dehydrated through a graded ethanol series and propylene oxide and embedded in LX112 maintaining a dorso-ventral orientation (Polysciences, Warrington, PA). Semithin (1 µm thick) sectioning was started at the frontal (cornea) pole of the embedded eye. Every 50 µm, sections were collected, stained with Toluidine blue and observed. Ultrathin sectioning was started when the presence of RPE was observed. Therefore, comparable regions of the eye, at each considered embryonal and post-partum stage, were analysed. Ultrathin sections were stained with uranyl acetate and lead citrate and analysed with EM10C or EM902A Zeiss electron microscopes.
HRP injection
In this study we used Oa1+/Y and Oa1–/Y mice from the same litter. At least six animals were used in each group. The age of the animals ranged from 4 to 5 months. All the animals were phenotypically black. Each animal was anaesthetized with 700 µl of Avertin. A single unilateral intraocular injection of HRP (30% HRP) was made into the left eye. The animals were allowed to recover. Approximately 24 h later they were deeply anaesthetized and perfused transcardially with 4% paraformaldehyde in PBS, pH 7.2. The brains were removed, postfixed in the perfusate for 2 h, washed in PBS and placed in 30% sucrose in PBS. The brains were frozen and sectioned at 40 µm in the coronal plane. A continuous series was collected from the optic tract through to the caudal pole of the SC in each animal. These sections were reacted to reveal the location of the anterogradely transported tracer along the visual pathway (33). The sections were mounted onto positively charged slides and air-dried. The sections were viewed under both bright-field and dark-field illumination. The borders of the LGN were identified by placing the bright-field condenser out of focus and/or with the aid of DIC optics. In a number of animals in which the label and nucleus were clear, the volume of the two was measured with the aid of a computer-based image analysis system. The threshold for frame-grabbed images was established at a level that identified the terminal label in the LGN in all of the animals examined. Consecutive images were grabbed through the full series of collected sections covering the LGN and the volume of the labelled region from the ipsilateral eye was calculated. It was not possible to repeat this process for the uncrossed projection to the SC, because even in the wild-type animals, the uncrossed projection to this region is divided into a number of relatively diffuse subregions.
Generation of melanocyte cultures
Primary cultures were made from trunk skin of Oa1+/+, Oa1+/– and Oa1 –/Y mice up to 24 h old, more or less as described previously (34,35). In short, the skin was split with trypsin, epidermal sheets were pooled and dissociated briefly with trypsin and EDTA and the resulting cell suspension was plated on to XB2 feeder cells. Generally 12 ml of culture (six 3 cm or three 5 cm dishes) were prepared per mouse. TPA and cholera toxin were added from the start of culture instead of day 4.
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ACKNOWLEDGEMENTS |
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The authors thank G. Borsani and M.T. Bassi, for critical reading of the manuscript; M. Smith for editorial help; E. Conti, C. Mocchetti, A. Orsi, E. Scanziani, L. Crippa, M. Cervia and K. Mai for constant technical assistance; U. Huffstadt for expert technical assistance; M. Matzuk for hprt- and tk-cassette vectors; A. Bradley for the AB2.2 ES cell line; W. Green for pictures of an OA1 patient RPE; and the instructors of the Molecular Embryology of the Mouse Course, CSHL 1995. This work was supported by grants from the Vision of Children; Telethon-Italy (grant no. E0942), CNR (target project ‘Biotechnology’) and MURST to C.T.; a Wellcome Trust grant to G. Jeffery; DFG grant SFB 430 C2 and fortuene grant no. 517.
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FOOTNOTES |
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+ To whom correspondence should be addressed. Tel: +39 081 5790113; Fax: +39 081 5790919; Email: incerti@tigem.it
§ Present address: Department of Pathology, Baylor College of Medicine, Houston, TX 70030, USA
¶ Present address: TIGEM, Via P. Castellino 11, I-80131 Naples, Italy
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REFERENCES |
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