ARTICLES Analysis of Ocular Hypopigmentation in Rab38cht/cht Mice Brian P. Brooks,1,2,3 Denise M. Larson,2,3 Chi-Chao Chan,1 Sten Kjellstrom,4 Richard S. Smith,5,6 Mary A. Crawford,1 Lynn Lamoreux,7 Marjan Huizing,3 Richard Hess,3 Xiaodong Jiao,1 J. Fielding Hejtmancik,1 Arvydas Maminishkis,1 Simon W. M. John,5,6 Ronald Bush,4 and William J. Pavan3 cht PURPOSE. To characterize the ocular phenotype resulting from and RPE thinning. The synergistic effects of the Rab38 and mutation of Rab38, a candidate gene for Hermansky-Pudlak Tyrp1b alleles suggest that TYRP1 is not the only target of syndrome. RAB38 trafficking. This mouse line provides a useful model for cht/cht METHODS. Chocolate mice (cht, Rab38 ) and control het- studying melanosome biology and its role in human ocular erozygous (Rab38cht/ϩ) and wild-type mice were examined diseases. (Invest Ophthalmol Vis Sci. 2007;48:3905–3913) clinically, histologically, ultrastructurally, and electrophysi- DOI:10.1167/iovs.06-1464 ologically. Mice homozygous for both the Rab38cht and the Tyrp1b alleles were similarly examined. he analysis of mice that exhibit defects in coat coloration cht/cht RESULTS. Rab38 mice showed variable peripheral iris trans- T(coat color mutants) has aided in the identification of 1 illumination defects at 2 months of age. Patches of RPE hypo- genes important in eye, skin, and hair pigmentation. Many of pigmentation were noted clinically in 57% of Rab38cht/cht eyes these genes are mutated in patients with pigmentary anoma- and 6% of Rab38cht/ϩ eyes. Rab38cht/cht mice exhibited thin- lies. Coat color mutants can exhibit a wide range of variation, ning of the iris and RPE and larger b-wave amplitudes in the including altered, dilute, or absent coloration. Skin and hair scotopic range when compared with the control animals. Com- coloration result from the pigment cells in hair follicles (mela- pared with wild-type mice, Rab38cht/cht melanosomes were nocytes) that synthesize a melanin-based pigment. Melanin is smaller and there were fewer in neuroectodermally derived also produced in the retinal pigment epithelium (RPE) and retinal pigment epithelium; in neural crest-derived choroid choroid of the eye. Melanocytes in skin, hair follicles, and melanocytes, they were smaller in size only. Mutation of both choroid are derived from the neural crest (NC), a transient Rab38 and Tyrp1 produced mice with ocular and coat color population of stem cells that arise early in development at the pigment dilution greater than that seen with either mutation dorsal neural tube. The RPE, in contrast, is derived from the alone. Comprehensive clinical and pathologic analyses showed neuroepithelium. no other organ system or blood defects in Rab38cht/cht mice. Melanin production occurs in specialized organelles within cht/cht pigment cells called melanosomes. The melanin synthetic en- CONCLUSIONS. Rab38 mice show ocular characteristics zymes (including tyrosinase, tyrosinase-related protein 1 reminiscent of human oculocutaneous albinism, as well as iris (TYRP1), and dopachrome tautomerase) must be trafficked within the melanocyte to the melanosome to achieve proper melanin production. Variations in the coat color of mouse From the 1National Eye Institute, the 3National Human Genome Research Institute, and the 4National Institute on Deafness and Other mutants can result from the absence of pigment cells (white Communication Disorders, National Institutes of Health, Bethesda, spots), defects in melanogenic enzymes, and aberrant traffick- Maryland; 5Howard Hughes Medical Institute, Bar Harbor, Maine; 6The ing of melanogenic proteins to the melanosome (color alter- Jackson Laboratory, Bar Harbor, Maine; and the 7Comparative Genetics ations or dilution). Program, TexasA&MUniversity, College Station, Texas. Genes mutated in mice with coat color variations have often 2Contributed equally to the work and therefore should be consid- been associated with human pigmentary anomalies with simi- ered equivalent authors. lar phenotypes. These include pigmentary glaucoma,2,3 oculo- Supported by the intramural program of the National Institutes of cutaneous and X-linked ocular albinism,4–7 Hermansky-Pudlak Health, the Howard Hughes Medical Institute, and National Eye Insti- syndrome (HPS),8 and Chediak-Higashi syndrome. HPS is an tute Grants EY01475 and EY11721. BPB is part of the Joint Physician- Scientist Development Program at the National Eye Institute and the autosomal recessive disease characterized by variable oculocu- National Human Genome Research Institute. taneous albinism (including foveal hypoplasia, nystagmus, and Submitted for publication December 11, 2006; revised April 30, iris transillumination defects), absent platelet-dense bodies 2007; accepted July 19, 2007. (leading to prolonged bleeding times), and sporadic lung fibro- Disclosure: B.P. Brooks, None; D.M. Larson, None; C.-C. Chan, sis.9,10 Genes for 16 hypopigmented mouse mutants with None; S. Kjellstrom, None; R.S. Smith, None; M.A. Crawford, None; platelet-mediated bleeding defects have been cloned, and mu- L. Lamoreux, None; M. Huizing, None; R. Hess, None; X. Jiao, tations in the human orthologous genes, most of which are None; J.F. Hejtmancik, None; A. Maminishkis, None; S.W.M. John, involved in organelle trafficking, were subsequently found in None; R. Bush, None; W.J. Pavan, None humans with HPS.11–13 The publication costs of this article were defrayed in part by page b/b charge payment. This article must therefore be marked “advertise- Similarly, Tyrp1 mice carry a mutation in a melanin ment” in accordance with 18 U.S.C. §1734 solely to indicate this fact. synthetic enzyme and exhibit a brown coat on a wild-type Corresponding author: Brian P. Brooks, National Eye Institute, black strain background. Patients with oculocutaneous albi- 6,14 National Institutes of Health, Building 10, Room 10B16, Bethesda, MD nism type 3 (OCA3) have homozygous TYRP1 mutations, 20892; [email protected]. which result in moderate hypopigmentation. Of course, there Investigative Ophthalmology & Visual Science, September 2007, Vol. 48, No. 9 Copyright © Association for Research in Vision and Ophthalmology 3905 Downloaded from tvst.arvojournals.org on 09/23/2021 3906 Brooks et al. IOVS, September 2007, Vol. 48, No. 9 can be human and mouse phenotype differences for disruption OH) and an indirect ophthalmoscope (Keeler, Windsor, Berkshire, UK) of the same gene. For example, mutation of Tyrp1 in both with a 90-D condensing lens (Volk, Mentor, OH). The mice were humans and mice results in hypopigmentation and iris transil- euthanatized with carbon dioxide according to institutional guidelines. lumination. However, DBA/2J mice homozygous for the “Young” mice were defined as 2 to 3 months of age; “aged” mice were Tyrp1b allele develop age-dependent iris stromal atrophy, ele- defined as older than 1 year. The fundi were also examined. vated intraocular pressure, and pigmentary glaucoma, which For body organ and blood system analysis, three male and three has not been observed in humans with TYRP1 mutations.2,3 female 5-month-old Rab38cht/cht mice were compared with six, age- This difference in known human and mouse phenotypes may and sex-matched wild-type control mice. We analyzed differences in be due to the nature of the mutation and whether or not the organ morphology, serum chemistries, hematocrit, and neutrophil and mutant TYRP1 protein can still stabilize tyrosinase. Because the platelet counts between Rab38cht/cht and wild-type mice. Age-related mouse iris atrophy phenotype is dependent on pigment pro- coat color changes were observed by visual comparison of subgroups duction and known human mutations induce ocular albinism, of mice. it has been suggested that the human mutations are self-rescu- These studies conformed to the principles for laboratory animal ing with respect to iris atrophy.2,3 research outlined by the Animal Welfare Act (National Institutes of The chocolate (Rab38cht/cht) mouse mutant arose sponta- Health/Department of Health and Human Services) and the ARVO neously on the C57BL/6J black background as a dark brown Statement for the Use of Animals in Ophthalmic and Vision Research coat color variant. A G19V point mutation in a highly con- and were approved by the Institutional Animal Care and Use Com- served amino acid of Rab38 is responsible for the chocolate mittee. phenotype.15,16 Rab38cht/cht mice have normal blood clotting times. In addition to the chocolate mouse, RAB38 is altered in Histopathology, Electron Microscopy, and a rat coat color mutant called Ruby (red-eyed dilution, R) Melanosome Image Analysis which has been proposed as an animal model for HPS.17 Ruby rats have hypopigmented eyes and coat and a bleeding diathe- For light microscopy, mouse eyes were enucleated and fixed in a phosphate-buffered paraformaldehyde–glutaraldehyde mixture, ac- sis. The Ruby Rab38 translation-initiation codon has a missense 22 mutation that is predicted to stop translation at the first codon cording to published protocols. Hematoxylin and eosin–stained and RAB38 protein is not detected. However, unlike in patients methacrylate-fixed sections from the pupillary–optic nerve axis were with HPS, platelet-dense granules are present with normal used for histopathology. appearance and numbers in Fawn-hooded hypertensive rats, For electron microscopy, mouse eyes were dissected and fixed in which also carry a first codon missense mutation in Rab38 and 4% glutaraldehyde in 0.15% phosphate buffer for 1 hour at room have a Ruby phenotype of hypopigmentation