Ординатура / Офтальмология / Английские материалы / Eye, Retina, and Visual System of the Mouse_Chalupa, Williams_2008

Figure 39.5 Radiation treatment prevents glaucomatous optic nerve excavation. A–F, Hematoxylin-eosin-stained sections of untreated and treated DBA/2J mouse eyes (A and B). Optic nerve head (ONH) (A) and retina (B) from nonglaucomatous DBA/2J mice showing large numbers of axons as evidenced by a thick nerve fiber layer (NFL) (arrowheads). ONH (C) and retina (D) of a treated DBA/2J mouse (14 months old) is indistinguishable from nonglaucomatous controls. E and F, In contrast, untreated DBA/2J mice show severe excavation of ONH (*) and no NFL. Cross sections of

radiation (such as atomic bomb survivors; Anderson et al., 2005). At this time, the mechanisms of neuroprotection are not known, and in its current form the treatment is not directly transferable to humans. Further studies designed, to elucidate the processes involved in radiation-induced neuroprotection have great potential for the development of powerful neuroprotective human therapies.

Genomics and Proteomics To improve our understanding of glaucoma and develop novel therapies, it is essential that the genes, proteins, and pathways involved in IOP elevation and glaucomatous optic nerve degeneration be identified. The human and mouse genome sequences are now available, and in functionally conserved units, such as proteincoding genes, they are more than 90% similar at the sequence level (Lander et al., 2001; Waterston et al., 2002). The majority of human genes have a mouse counterpart, and therefore the identification of genes that play a role in glaucoma in mice may provide important insight into mechanisms involved in human glaucoma.

Microarray-based technologies enable glaucoma-relevant tissues to be probed for genes that change in response to IOP elevation and those involved in RGC death and axon degeneration. In the first study of its kind in DBA/2J mice, microarray data have provided insight into pathways likely to be activated downstream of IOP elevation. RNA isolated from retinas from 8-month-old DBA/2J mice that had received a

the optic nerve from treated (G) and untreated (H) DBA/2J mice stained with PPD, a stain that labels myelin sheath and sick or dying axons. The severe axon loss and scarring in the untreated eye are absent in the treated eye. The vast majority (>96%) of eyes from radiation-treated DBA/2J mice were completely rescued from glaucoma. See color plate 35. (Modified from Anderson et al., 2005. Reproduced from Proceedings of the National Academy of Sciences of the USA, 2005, 102:4566–4571. Copyright © 2005 National Academy of Sciences.)

pressure insult was compared with RNA from 3-month-old control retinas. Sixty-eight genes showed differences in their expression, including genes involved in glial activation (e.g., ceruloplasmin and glial fibrillary acidic protein) and immune responses (e.g., lipocalin) (Steele et al., 2006). These findings are in general agreement with experiments carried out in the rat, but further work is needed to determine whether these are primary causes or a consequence of an earlier glaucomatous insult.

As a complement to microarray-based approaches, proteomic analyses are uncovering proteins and pathways involved in glaucoma. Peptidyl arginine deiminase (PAD2), an enzyme that converts protein arginine to citrulline, was found only in samples taken from human POAG patients, and not in samples from unaffected individuals (Bhattacharya et al., 2006). This difference suggests a role for citrullination and structural disruption of myelination in glaucoma and can be interrogated further using mouse models.

Mutagenesis and new models

Glaucoma in humans is a complex multifactorial disease. DBA/2J is currently the most utilized mouse model of glaucoma and has many of the hallmarks of human glaucoma, including a multifactorial and complex etiology. Nevertheless, because glaucoma is a manifestation of a heterogeneous group of complex processes, it is important to develop alter-

native models of other forms of inherited glaucoma and on strains with distinct genetic backgrounds. POAG is the most common form of glaucoma, and models with altered genes shown to cause POAG in humans have been made (e.g., MYOC, discussed earlier). However, no convincing mouse model of POAG, with IOP elevation and optic nerve excavation, has been reported. An important strategy for producing such models involves the random mutagenesis of the mouse genome. Since the genes underlying these new models can be identified, this approach simultaneously provides valuable resources for identifying new causative pathways of IOP elevation. Commonly, the genomes of founder males are mutagenized with chemical agents such as ethyl nitrosourea, and breeding and screening strategies are developed to uncover phenotypes of interest (Thaung et al., 2002). This type of strategy has proved successful in other complex diseases and is crucial for full exploitation of the strengths of the mouse system to understand the diverse causes of glaucoma.

Conclusion

The genetic networks that determine glaucoma susceptibility are still largely undefined. Many components remain to be identified, and the ways in which known genes interact with other genes are not well characterized. Genetic studies in the mouse have yielded fundamental insights into glaucoma. Aggressive use of model systems and new advances in genomics, proteomics, and genetic analysis will make mouse models even more valuable. Mouse studies are an important complement to those in humans and other species. Only by integrating information from diverse sources will we gain an in-depth understanding of the pathogenesis of glaucoma and begin to develop improved glaucoma therapies.

REFERENCES

Ahmed, E., Ma, J., Rigas, I., Hafezi-Moghadam, N., Iliaki, E., Gragoudas, E. S., Miller, J. W., and Adamis, A. P. (2003). Non-invasive tonometry in the mouse. Invest. Opthalmol. Vis. Sci. 44:3336.

Aihara, M., Lindsey, J. D., and Weinreb, R. N. (2003a). Experimental mouse ocular hypertension: Establishment of the model.

Invest. Ophthalmol. Vis. Sci. 44(10):4314–4320.

Aihara, M., Lindsey, J. D., and Weinreb, R. N. (2003b). Ocular hypertension in mice with a targeted type I collagen mutation.

Invest. Ophthalmol. Vis. Sci. 44(4):1581–1585.

Anderson, M. G., Libby, R. T., Gould, D. B., Smith, R. S., and John, S. W. (2005). High-dose radiation with bone marrow transfer prevents neurodegeneration in an inherited glaucoma.

Proc. Nat. Acad. Sci. U.S.A. 102(12):4566–4571.

Anderson, M. G., Libby, R. T., Mao, M., Cosma, I. M., Wilson, L. A., Smith, R. S., and John, S. W. (2006). Genetic context determines susceptibility to intraocular pressure elevation in a mouse pigmentary glaucoma. BMC Biol. 4:20.

Anderson, M. G., Smith, R. S., Hawes, N. L., Zabaleta, A., Chang, B., Wiggs, J. L., and John, S. W. (2002). Mutations in genes encoding melanosomal proteins cause pigmentary glaucoma in DBA/2J mice. Nat. Gen. 30(1):81–85.

Avila, M. Y., Carre, D. A., Stone, R. A., and Civan, M. M. (2001). Reliable measurement of mouse intraocular pressure by a servo-null micropipette system. Invest. Ophthalmol. Vis. Sci. 42 (8):1841–1846.

Bhattacharya, S. K., Crabb, J. S., Bonilha, V. L., Gu, X., Takahara, H., and Crabb, J. W. (2006). Proteomics implicates peptidyl arginine deiminase 2 and optic nerve citrullination in glaucoma pathogenesis. Invest. Ophthalmol. Vis. Sci. 47(6):2508– 2514.

Bhattacharya, S. K., Peachey, N. S., and Crabb, J. W. (2005a). Cochlin and glaucoma: A mini-review. Vis. Neurosci. 22(5):605– 613.

Bhattacharya, S. K., Rockwood, E. J., Smith, S. D., Bonilha, V. L., Crabb, J. S., Kuchtey, R. W., Robertson, N. G., Peachey, N. S., Morton, C. C., et al. (2005b). Proteomics reveal cochlin deposits associated with glaucomatous trabecular meshwork. J. Biol. Chem. 280(7):6080–6084.

Chang, B., Smith, R. S., Hawes, N. L., Anderson, M. G., Zabaleta, A., Savinova, O., Roderick, T. H., Heckenlively, J. R., Davisson, M. T., et al. (1999). Interacting loci cause severe iris atrophy and glaucoma in DBA/2J mice. Nat. Genet. 21(4): 405–409.

Dalke, C., Pleyer, U., and Graw, J. (2005). On the use of TonoPen XL for the measurement of intraocular pressure in mice. Exp. Eye Res. 80(2):295–296.

Danias, J., Kontiola, A. I., Filippopoulos, T., and Mittag, T. (2003a). Method for the noninvasive measurement of intraocular pressure in mice. Invest. Ophthalmol. Vis. Sci. 44(3):1138–1141.

Danias, J., Lee, K. C., Zamora, M. F., Chen, B., Shen, F., Filippopoulos, T., Su, Y., Goldblum, D., Podos, S. M., et al. (2003b). Quantitative analysis of retinal ganglion cell (RGC) loss in aging DBA/2NNia glaucomatous mice: Comparison with RGC loss in aging C57/BL6 mice. Invest. Ophthalmol. Vis. Sci. 44(12):5151–5162.

Filippopoulos, T., Matsubara, A., Danias, J., Huang, W., Dobberfuhl, A., Ren, L., Mittag, T., Miller, J. W., and Grosskreutz, C. L. (2006). Predictability and limitations of non-invasive murine tonometry: Comparison of two devices. Exp. Eye Res. 83(1):194–201.

Goldblum, D., and Mittag, T. (2002). Prospects for relevant glaucoma models with retinal ganglion cell damage in the rodent eye. Vision Res. 42(4):471–478.

Gould, D. B., and John, S. W. (2002). Anterior segment dysgenesis and the developmental glaucomas are complex traits. Hum. Mol. Genet. 11(10):1185–1193.

Gould, D. B., Miceli-Libby, L., Savinova, O. V., Torrado, M., Tomarev, S. I., Smith, R. S., and John, S. W. (2004). Genetically increasing Myoc expression supports a necessary pathologic role of abnormal proteins in glaucoma. Mol. Cell. Biol. 24(20): 9019–9025.

Gould, D. B., Reedy, M., Wilson, L. A., Smith, R. S., Johnson, R. L., and John, S. W. (2006). Mutant myocilin nonsecretion in vivo is not sufficient to cause glaucoma. Mol. Cell. Biol. 26(22):8427– 8436.

Gould, D. B., Smith, R. S., and John, S. W. (2004). Anterior segment development relevant to glaucoma. Int. J. Dev. Biol. 48(8–9):1015–1029.

Gross, R. L., Ji, J., Chang, P., Pennesi, M. E., Yang, Z., Zhang, J., and Wu, S. M. (2003). A mouse model of elevated intraocular

pressure: Retina and optic nerve findings. Trans. Am. Ophthalmol. Soc. 101:163–169 [discussion 169–171].

Grozdanic, S. D., Betts, D. M., Sakaguchi, D. S., Allbaugh, R. A., Kwon, Y. H., and Kardon, R. H. (2003) Laser-induced mouse model of chronic ocular hypertension. Invest. Ophthalmol. Vis. Sci. 44(10):4337–4346.

Huang, W., Fileta, J. B., Dobberfuhl, A., Filippopolous, T., Guo, Y., Kwon, G., and Grosskreutz, C. L. (2005). Calcineurin cleavage is triggered by elevated intraocular pressure, and calcineurin inhibition blocks retinal ganglion cell death in experimental glaucoma. Proc. Nat. Acad. Sci. U.S.A. 102(34):12242– 12247.

Jakobs, T. C., Libby, R. T., Ben, Y., John, S. W., and Masland, R. H. (2005). Retinal ganglion cell degeneration is topological but not cell type specific in DBA/2J mice. J. Cell. Biol. 171 (2):313–325.

John, S. W. (2005). Mechanistic insights into glaucoma provided by experimental genetics. The Cogan Lecture. Invest. Ophthalmol. Vis. Sci. 46(8):2650–2661.

John, S. W., Anderson, M. G., and Smith, R. S. (1999). Mouse genetics: A tool to help unlock the mechanisms of glaucoma. J. Glaucoma 8(6):400–412.

John, S. W., Hagaman, J. R., MacTaggart, T. E., Peng, L., and Smithes, O. (1997). Intraocular pressure in inbred mouse strains.

Invest. Ophthalmol. Vis. Sci. 38(1):249–253.

John, S. W., Smith, R. S., Savinova, O. V., Hawes, N. L., Chang, B., Turnbull, D., Davisson, M., Roderick, T. H., and Heckenlively, J. R. (1998). Essential iris atrophy, pigment dispersion, and glaucoma in DBA/2J mice. Invest. Ophthalmol. Vis. Sci. 39(6):951–962.

Kanagavalli, J., Pandaranayaka, E., Krishnadas, S. R., Krishnaswamy, S., and Sundaresan, P. (2004). A review of genetic and structural understanding of the role of myocilin in primary open angle glaucoma. Ind. J. Ophthalmol. 52(4):271– 280.

Kim, B. S., Savinova, O. V., Reedy, M. V., Martin, J., Lun, Y., Gan, L., Smith, R. S., Tomarev, S. I., John, S. W., et al. (2001). Targeted disruption of the myocilin gene (Myoc) suggests that human glaucoma-causing mutations are gain of function. Mol. Cell. Biol. 21(22):7707–7713.

Kroeber, M., Ohlmann, A., Russell, P., and Tamm, E. R. (2006). Transgenic studies on the role of optineurin in the mouse eye. Exp. Eye Res. 82(6):1075–1085.

Lander, E. S., Linton, L. M., Birren, B., Nusbaum, C., Zody, M. C., Baldwin, J., Devon, K., Dewar, K., Doyle, M., et al. (2001). Initial sequencing and analysis of the human genome. Nature 409(6822):860–921.

Leske, M. C. (1983). The epidemiology of open-angle glaucoma: A review. Am. J. Epidemiol. 118(2):166–191.

Libby, R. T., Anderson, M. G., Pang, I. H., Robinson, Z. H., Savinova, O. V., Cosma, I. M., Snow, A., Wilson, L. A., Smith, R. S., et al. (2005a). Inherited glaucoma in DBA/2J mice: Pertinent disease features for studying the neurodegeneration. Vis. Neurosci. 22(5):637–648.

Libby, R. T., Gould, D. B., Anderson, M. G., and John, S. W. (2005b). Complex genetics of glaucoma susceptibility. Annu. Rev. Genom. Hum. Genet. 6:15–44.

Libby, R. T., Li, Y., Savinova, O. V., Barter, J., Smith, R. S., Nickells, R. W., and John, S. W. (2005c). Susceptibility to neurodegeneration in a glaucoma is modified by Bax gene dosage. PLoS Genet. 1(1):17–26.

Libby, R. T., Smith, R. S., Savinova, O. V., Zabaleta, A., Martin, J. E., Gonzalez, F. J., and John, S. W. (2003). Modifi-

cation of ocular defects in mouse developmental glaucoma models by tyrosinase. Science 299(5612):1578–1581.

Lindsey, J. D., and Weinreb, R. N. (2002). Identification of the mouse uveoscleral outflow pathway using fluorescent dextran.

Invest. Ophthalmol. Vis. Sci. 43(7):2201–2205.

Lindsey, J. D., and Weinreb, R. N. (2005). Elevated intraocular pressure and transgenic applications in the mouse. J. Glaucoma 14(4):318–320.

Liu, Y., and Vollrath, D. (2004). Reversal of mutant myocilin non-secretion and cell killing: Implications for glaucoma. Hum. Mol. Gene. 13(11):1193–1204.

Mabuchi, F., Aihara, M., Mackey, M. R., Lindsey, J. D., and Weinreb, R. N. (2004). Regional optic nerve damage in experimental mouse glaucoma. Invest. Ophthalmol. Vis. Sci. 45(12): 4352–4358.

May, C. A., and Lutjen-Drecoll, E. (2002). Morphology of the murine optic nerve. Invest. Ophthalmol. Vis. Sci. 43(7):2206–2212.

May, C. A., and Mittag, T. (2006). Optic nerve degeneration in the DBA/2NNia mouse: Is the lamina cribrosa important in the development of glaucomatous optic neuropathy? Acta Neuropathol. 111(2):158–167.

McKinnon, S. J., Reitsamer, H. A., Ransom, N. L., Caldwell, M., Harrison, J. M., and Kiel, J. W. (2003). Induction and TonoPen measurement of ocular hypertension in C57BL/6 Mice. ARVO Abstract.

McMenamin, P. G. (1999). Dendritic cells and macrophages in the uveal tract of the normal mouse eye. Br. J. Ophthalmol. 83(5): 598–604.

McMenamin, P. G., and Holthouse, I. (1992). Immunohistochemical characterization of dendritic cells and macrophages in the aqueous outflow pathways of the rat eye. Exp. Eye Res. 55 (2):315–324.

Mo, J. S., Anderson, M. G., Gregory, M., Smith, R. S., Savinova, O. V., Serreze, D. V., Ksander, B. R., Streilein, J. W., and John, S. W. (2003). By altering ocular immune privilege, bone marrow-derived cells pathogenically contribute to DBA/2J pigmentary glaucoma. J. Exp. Med. 197(10):1335–1344.

Morris, C. A., Crowston, J. G., Lindsey, J. D., Danias, J., and Weinreb, R. N. (2006). Comparison of invasive and noninvasive tonometry in the mouse. Exp. Eye Res. 82(6):1094–1099.

Morrison, J. C., Moore, C. G., Deppmeier, L. M., Gold, B. G., Meshul, C. K., and Johnson, E. C. (1997). A rat model of chronic pressure-induced optic nerve damage. Exp. Eye Res. 64(1):85–96.

Pease, M. E., Hammond, J. C., and Quigley, H. A. (2006). Manometric calibration and comparison of TonoLab and TonoPen tonometers in rats with experimental glaucoma and in normal mice. J. Glaucoma 15(6):512–519.

Petros, T. J., Williams, S. E., and Mason, C. A. (2006). Temporal regulation of EphA4 in astroglia during murine retinal and optic nerve development. Mol. Cell. Neurosci. 32(1–2):49–66.

Quigley, H. A. (1995). Ganglion cell death in glaucoma: Pathology recapitulates ontogeny. Aust. N.Z. J. Ophthalmol. 23(2):85–91.

Quigley, H. A. (1996). Number of people with glaucoma worldwide. Br. J. Ophthalmol. 80(5):389–393.

Quigley, H. A., Hohman, R. M., Addicks, E. M., Massof, R. W., and Green, W. R. (1983). Morphologic changes in the lamina cribrosa correlated with neural loss in open-angle glaucoma. Am. J. Ophthalmol. 95(5):673–691.

Reichstein, D., Ren, L., Filippopoulos, T., Mittag, T., and Danias, J. (2007). Apoptotic retinal ganglion cell death in the DBA/2 mouse model of glaucoma. Exp. Eye Res. 84(1): 13–21.

Reitsamer, H. A., Kiel, J. W., Harrison, J. M., Ransom, N. L., and McKinnon, S. J. (2004). TonoPen measurement of intraocular pressure in mice. Exp. Eye Res. 78(4):799–804.

Rezaie, T., Child, A., Hitchings, R., Brice, G., Miller, L., Coca-Prados, M., Heon, E., Krupin, T., Ritch, R., et al. (2002). Adult-onset primary open-angle glaucoma caused by mutations in optineurin. Science 295(5557):1077–1079.

Ritch, R., Shields, M. B., and Krupin, T. (1996). The glaucomas: Clinical science, 2nd ed. St Louis: Mosby–Year Book.

Savinova, O. V., Sugiyama, F., Martin, J. E., Tomarev, S. I., Paigen, B. J., Smith, R. S., and John, S. W. (2001). Intraocular pressure in genetically distinct mice: An update and strain survey.

BMC Genet. 2:12.

Schlamp, C. L., Li, Y., Dietz, J. A., Janssen, K. T., and Nickells, R. W. (2006). Progressive ganglion cell loss and optic nerve degeneration in DBA/2J mice is variable and asymmetric. BMC Neurosci. 7:66.

Scott, T. S., and Wirtz, M. K. (1996). Biochemistry of aqueous outflow. In R. Ritch, M. B. Shields, and T. Krupin (Eds.), The glaucomas, 2nd ed. (vol. 1, pp. 281–305). St Louis: Mosby.

Senatorov, V., Malyukova, I., Fariss, R., Wawrousek, E. F., Swaminathan, S., Sharan, S. K., and Tomarev, S. (2006). Expression of mutated mouse myocilin induces open-angle glaucoma in transgenic mice. J. Neurosci. 26(46):11903–11914.

Shields, M. B. (1997). Shields’ textbook of glaucoma, 4th ed. Baltimore: Williams and Wilkins.

Steele, M. R., Inman, D. M., Calkins, D. J., Horner, P. J., and Vetter, M. L. (2006). Microarray analysis of retinal gene expression in the DBA/2J model of glaucoma. Invest. Ophthalmol. Vis. Sci. 47(3):977–985.

Strom, R. C., and Williams, R. W. (1998). Cell production and cell death in the generation of variation in neuron number. J. Neurosci. 18(23):9948–9953.

Thaung, C., West, K., Clark, B. J., McKie, L., Morgan, J. E., Arnold, K., Nolan, P. M., Peters, J., Hunter, A. J., et al. (2002). Novel ENU-induced eye mutations in the mouse: Models for human eye disease. Hum. Mol. Genet. 11(7):755–767.

Vincent, A. L., Billingsley, G., Buys, Y., Levin, A. V., Priston, M., Trope, G., Williams-Lyn, D., and Heon, E. (2002). Digenic inheritance of early-onset glaucoma: CYP1B1, a potential modifier gene. Am. J. Hum. Genet. 70(2):448–460.

Wang, W. H., Millar, J. C., Pang, I. H., Wax, M. B., and Clark, A. F. (2005). Noninvasive measurement of rodent intraocular pressure with a rebound tonometer. Invest. Ophthalmol. Vis. Sci. 46(12):4617–4621.

Waterston, R. H., Lindblad-Toh, K., Birney, E., Rogers, J., Abril, J. F., Agarwal, P., Agarwala, R., Ainscough, R., Alexandersson, M., et al. (2002). Initial sequencing and comparative analysis of the mouse genome. Nature 420(6915): 520–562.

Weinreb, R. N., and Lindsey, J. D. (2005). The importance of models in glaucoma research. J. Glaucoma 14(4):302–304.

Willoughby, C. E., Chan, L. L., Herd, S., Billingsley, G., Noordeh, N., Levin, A. V., Buys, Y., Trope, G., Sarfarazi, M., et al. (2004). Defining the pathogenicity of optineurin in juvenile open-angle glaucoma. Invest. Ophthalmol. Vis. Sci. 45(9): 3122–3130.

Wu, H., Byrne, M. H., Stacey, A., Goldring, M. B., Birkhead, J. R., Jaenisch, R., and Krane, S. M. (1990). Generation of collagenase-resistant collagen by site-directed mutagenesis of murine pro alpha 1(I) collagen gene. Proc. Nat. Acad. Sci. U.S.A. 87(15):5888–5892.

Zillig, M., Wurm, A., Grehn, F. J., Russell, P., and Tamm, E. R. (2005). Overexpression and properties of wild-type and Tyr437His mutated myocilin in the eyes of transgenic mice.

Invest. Ophthalmol. Vis. Sci. 46(1):223–234.

Cataracts as lens opacities are associated with a group of well-known diseases that are particularly common in the elderly population. In contrast to age-related forms of cataract, congenital cataracts, or cataracts that develop in early childhood, are rather rare and occur in developed countries with a frequency of around 30 cases per 100,000 births, with another 10 cases being diagnosed by the age of 15 years (mainly as dominant forms). Rates are likely to be higher in developing countries because of rubella infections and consanguinity (for the recessive forms; Gibert et al., 2003).

In recent years, mice have proved to be excellent models for ophthalmologists, because the clinical phenotypes are quite similar to human conditions. The first systematic evaluation of large mouse populations for mutations affecting the eye lens at birth was initiated in 1979, when Kratochvilova and Ehling described screening for murine dominant cataract mutants in the F1 generation after paternal radiation treatment. Systematic screening for eye mutants was extended to the use of ethylnitrosourea (ENU) as a mutagenic agent (Ehling et al., 1985), and to the targeted inactivation of genes. In this chapter, I review mutant mouse strains in which the lens is affected early in embryonic development or later in life, resulting in senile cataracts. Because lens development and the developmental genetics of the eye and retina are treated elsewhere in this book (see chapters 22 and 24), only the most important aspects are discussed here. The large number of transgenic mice strains that overexpress various genes in the lens also are excluded from this chapter’s discussion because the ectopic expression of a gene in the lens frequently leads to cataracts. The interested reader is directed to previously published reviews with many original citations (Graw, 2003, 2004). The chromosomal position of genes and their corresponding mutations can be found at the Web site of the Jackson Laboratory (www. informatics.jax.org/; menu, “Genes and Markers”).

Mutations at early stages of lens development

PAX6 and PITX3 One of the central genes in eye development is the paired-box gene Pax6, which was recognized as being affected in the mouse small eye (Sey) mutants (Hill et al., 1991). Pax6 maps at mouse chromosome 2; the actual list of the Jackson Laboratory (January 2008) contains 28 alleles in the mouse, of which four are targeted mutations. In the classic Sey mutant, the failure in lens development is attributed to

a defect in the inductive interaction between the optic vesicle and the overlying ectoderm, since these tissues fail to make discrete contacts. A typical example is shown in figure 40.1A. Homozygotes die around the time of birth because of breathing problems.

The expressivity of heterozygous Pax6 mutations is variable, with mutants expressing a range of phenotypes from small anterior polar cataracts to the more extreme phenotype of anterior polar opacity, corneal adhesions, iris abnormalities, and microphthalmia. The morphological alterations correspond to the expression pattern of Pax6. Pax6 transcripts are first detected in the presumptive foreand hindbrain of 8-day-old mouse embryos; at E8.5 it is present in the optic sulcus, the lateral evagination at the basis of the forebrain. Later, at E9.5, Pax6 is expressed in the optic vesicle, the optic stalk, and the surface ectoderm, which will give rise to the lens. Between E10 and E12, Pax6 is observed in the inner layer of the optic cup, in the lens, and in the surface ectoderm, which at this stage gives rise to the future cornea. In the elongating primary fiber cells, Pax6 has a posterior localization. At E15.5, Pax6 is expressed in the two layers of the neural retina, the anterior epithelium of the cornea, and the lens. Besides the eye, Pax6 occurs in specific regions of the brain, the olfactory epithelium, and the pancreas (for a review, see Graw, 2004). In particular, mouse Pax6 mutants exhibit changes in neurogenesis, cell proliferation, and patterning in the brain (Haubst et al., 2004; Graw et al., 2005).

The second interesting gene in the context of early lens development is Pitx3. In the mouse mutant aphakia (ak), the promoter of the Pitx3 gene is affected by two deletions (Semina et al., 2000; Rieger et al., 2001). The phenotype is characterized at early stages of development by a small lens vesicle with a stable contact to the cornea, the lens stalk. In later stages the lens vesicle is degraded, which leads to the formation of a lensless eye, giving this mutant its name. This feature is shown in figure 40.1B. Another mutant line, Cat4a, shares one aspect with the aphakia mutant, the inhibition of the separation of the lens vesicle from the surface ectoderm (Grimes et al., 1998). However, Cat4a is mapped to mouse chromosome 8, suggesting that it is different from Pitx3.

The phenotype of the mouse mutant correlates well with the expression pattern of the affected gene Pitx3. It is strongly expressed in the developing lens vesicle starting at E11, but later also throughout the lens, particularly in the anterior

Figure 40.1 Mutations in transcription factors and their effect on lens development. A, Histological section through a developing eye of a Pax6 mutant (Pax6Aey11; E17.5) clearly demonstrates the persisting lens stalk (arrow), the connection between the lens and the cornea. B, Histological section of the developing eye of the aphakia

epithelium and equator region. Moreover, there are recent reports that Pitx3 is also expressed in the dopaminergic neurons of the substantia nigra in the brain. It is not surprising, then, that aphakia mice also exhibit a selective loss of these particular neurons (Hwang et al., 2003) and a malformation of the mesencephalic dopamine system (Smidt et al., 2004).

MAF, FOX, and SOX Other genes coding for transcription factors important for eye and lens development include Maf,

Sox1, Sox2, FoxC1, and FoxE3. In particular, Maf and Sox1 act as transcription factors on the promoters of the γ- crystallin-encoding genes (Cryg).

The Fox transcription factors are characterized by a 110-amino acid motif originally defined as a DNA-binding domain in the Drosophila transcription factor forkhead (Fox: forkhead box). A mutation in FoxE3 was shown to cause the phenotype in the mouse mutant dysgenic lens (dyl), first published in 1979. In this mutant, the lens vesicle fails to separate from the ectoderm, causing the lens and the cornea to fuse; the dyl phenotype includes loss of lens epithelium; a small, cataractous lens; and malformations of most tissues of the anterior segment (iris, cornea, ciliary body, and trabecular meshwork; Blixt et al., 2006). Moreover, a mutation in the mouse Foxc1 mutant led to a similar phenotype with additional glaucoma (Hong et al., 1999).

The Maf family of basic region leucine zipper (bZIP) transcription factors was first identified through the v-maf oncogene, an avian retrovirus-transforming gene. The targeted deletion of c-Maf in the mouse leads to a stop of lens primary fiber cell elongation at the lens vesicle stage (Ring

mouse (deletions in the Pitx3 promoter) shows absence of the lens at E18.5; the eyeball is filled with retinal derivatives. C, cornea; R, retina. See color plate 36. (A, From Graw et al., 2005. B, From Semina et al., 2000.)

et al., 2000); the same feature was published for a mild pulverulent cataract mutant in mouse (opaque flecks in the lens, Opj; Lyon et al., 2003). The point mutation affects the basic region of the DNA-binding domain. In general, Maf binds as homoor heterodimer to so-called Maf-responsive elements (MAREs). MAREs are found in the promoters of the crystallin-encoding genes; Maf itself is upregulated by Pax6 (Civil et al., 2002).

The Sox family of transcription factors has an HMG domain (high mobility group) in common; the founder of this family is the Sry gene (sex-determining region of Y chromosome). The genes Sox1, Sox2, and Sox3 are expressed in the mouse in the CNS and in the sensory placodes. In particular, Sox2 is expressed during early eye development in the lens placode in the portion of the ectoderm that is in contact with the optic cup and invaginates to form the lens vesicle. This invagination coincides with the onset of Sox1 expression in the mouse lens placode. At later stages, Sox2 is downregulated and Sox1 is upregulated (Kamachi et al., 2000).

A targeted deletion of Sox1 in mice causes microphthalmia and cataract. Mutant lens fiber cells fail to elongate, probably as a result of an almost complete absence of Cryg transcripts (Nishiguchi et al., 1998). The phenotype of the homozygous Sox1 deletion mutant is very similar to the most severe Cryg mutation, Cryget. In contrast to Sox1, mutations in the human SOX2 gene cause anophthalmia (without cataract; Fantes et al., 2003), however, the heterozygous knockout mice of Sox2 appeared normal, but the homozygous mutants are peri-implantationally lethal (Avilion et al., 2003).

Miscellaneous Several other genes (e.g., Shh, rx/eyeless, Lhx, Bmp4, Bmp7) are known to be expressed at these early stages; however, the phenotypes of the corresponding knockout or null mutants manifest mainly with anophthalmia (loss of the entire ocular structure) or microphthalmia (small eye), but no cataracts. For a further discussion of these genes, readers may refer to previous reviews and references therein (e.g., Graw, 2003; see also chapter 22, this volume).

After the lens placode stage, the next important step is the formation of the lens vesicle. Its key role is addressed by some mouse mutants, such as extra toes (Gli3Xt: Franz and Besecke, 1991), eye lens aplasia (elap: Aso et al., 2001), and some “blebbing” mutants (bl, my, eb, heb). All these mouse models have in common a microphthalmic phenotype with major disturbances in most of the ocular tissues. The lens is frequently missing; correspondingly, these mutations are not associated with cataracts. The blebbing mutants are characterized by mutations in genes affecting extracellular matrix proteins (Kiyozumi et al., 2006). For the Gli3 mutant, an interesting increase in the penetrance of the ocular phenotype (including lens defects) was reported if it was crossed with a Pax6+/− background (Zaki et al., 2006).

Mutations affecting the lens membranes

As discussed earlier, in only a few cases are cataracts formed at the early stages of eye development. However, one might assume that more and diverse phenotypes of cataracts would occur if stages are affected when the lens vesicle is already formed. This section describes mutants that show defects in the lens cell membrane.

Aquaporin/Mip One of the first detected cataract mutations was the Fraser cataract (CatFr; Fraser and Schabtach, 1962). In this mutant, the cell nuclei in the deep cortex become abnormally pyknotic (beginning at E14); degeneration of cytoplasm and destruction of the lenticular nucleus follow. CatFr was shown to be allelic with another mouse mutant, referred to as lens opacity (Lop). The two alleles, CatLop and CatFr, were mapped 20 cM distal to steel (Sl) on chromosome 10. A candidate gene for the Cat locus encodes the membrane intrinsic protein (gene symbol: Mip), and sequence analysis finally revealed that the CatFr mutation is due to a transposoninduced splicing error leading to a truncated form of Mip transcripts. The other allele, CatLop (G151C), leads to a single amino acid substitution (Ala51Pro), which inhibits targeting of Mip to the cell membrane (Shiels and Bassnett, 1996). In total, seven phenotypic alleles have been reported (MGI database, December 2006); in general, homozygous mutants have microphthalmia and lens opacity. Other defects may include degeneration of lens fiber cells, vacuolization of lens fibers, and a reduced γ- to α-crystallin ratio. Heterozygotes have less severe forms of lens cataract and microphthalmia.

Mip forms specialized junctions between the fiber cells and can be first detected in the primary fiber cells of the early lens vesicle. In situ hybridization demonstrated that Mip expression is highest in the elongating fiber cells in the bow region of the lens; Mip antiserum specifically decorates fiber cell membranes, highlighting their regular anterior to posterior organization. Mip is also referred to aquaporin-0; a review on the role of aquaporin water channels in eye function was published a few years ago (Verkman, 2003). Recently, an Aqp1-knockout mutation was reported. These mice do not exhibit lens opacification; however, if these mutants are treated first with 3-methylcholantrene and later with acetaminophen, all mice tested develop cataract, indicating a complex interaction of environmental factors (chemical treatment) and genetic constitution (Ruiz-Ederra and Verkman, 2006).

LIM2 The total opacity (To3 ) gene mutation is placed on chromosome 7; mice heterozygous or homozygous for the To3 mutation exhibit a total opacity of the lens with a dense cataract. Additionally, homozygotes exhibit microphthalmia and abnormally small eyes. Histological analysis revealed vacuolization of the lens and gross disorganization of the fibers; posterior lens rupture can be observed only in homozygotes. The To3 mutation was characterized as a single G→T transversion within the first exon of the Lim2 gene coding for a lens-specific integral membrane protein, MP19. It was predicted that this DNA change would result in a nonconservative substitution (Gly15Val; Steele et al., 1997). In the mutants, MP19 is not transported to the lens fiber cell membranes but appears to be trapped in a subcellular compartment within the cells (Chen et al., 2002).

Connexins in the Lens Lens fiber cells are coupled by intercellular gap junction channels, particularly by the connexins 46 and 50 (also known as MP70). Since they are referred to as α3 or α8 subunits, their gene symbols are Gja3 and Gja8, respectively. Connexins have four transmembrane domains with three intracellular regions (the N-terminus, a cytoplasmatic loop, and the C-terminus) and two extracellular loops. Six connexin subunits oligomerize to form one hemichannel; the entire channel is formed by the docking of extracellular loops of two opposing hemichannels. The presence of two types of subunits in a cell allows the formation of a broad variety of channels.

Gja3 is mapped to mouse chromosome 14; a knockout mutation of Gja3 exhibits nuclear cataract, which was associated with the proteolysis of crystallins. Obviously, there is no influence on the early stages of lens formation (Gong et al., 1997). No other mouse Gja3 mutation has been described to date. A mutation in the human GJA3 gene results in an amino acid substitution in the first transmembrane region of

connexin 46. The mutation was shown to be causative of a congenital nuclear pulverulent cataract ( Jiang et al., 2003); however, a few years ago, Rees et al. (2000) and Mackay et al. (1999) showed that mutations in the human GJA3 gene also led to zonular pulverulent cataracts. The phenotypic differences might be explained by the fact that they affect different parts of the protein (the extracellular and intracellular domain).

In contrast to Gja3, actually seven cataract-causing alleles of the Gja8 gene are reported. Gja8 maps to mouse chromosome 3 and was demonstrated to be affected by a single A→C transversion within codon 47 of the No2 (nuclear opacity 2) mouse cataract. The sequence alteration is predicted to result in the nonconserved substitution of Ala for the normally encoded Asp (Steele et al., 1998). A similar phenotype (microphthalmia and nuclear cataract) was observed in Cx50 null mice (White et al., 1998). Typical examples of isolated lenses from Gja8 mutant mice are shown in figure 40.2A. A recent article on Gja3 and Gja8 mutations discusses in detail the role of the corresponding connexins during lens fiber cell formation (Xia et al., 2006b).

Mutations affecting the structural proteins of the lens

Up to 90% of the soluble protein in the postmitotic lens cells consists of proteins, which are referred to as α-, β-, and γ- crystallins (Mörner, 1893). The α-crystallins form high- molecular-weight aggregates formed by both, αAand

A B

Figure 40.2 Mutations in Gja8 and Cryaa. Gross appearance of lenses of the Gja8Aey5 mutant mouse (A) compared with lenses from a CryaaAey7 mutant mouse (B). The upper row indicates the phenotype for the heterozygotes and the lower row that for the homozygous mutants. In both cases, a gene dose effect can be observed. See color plate 37.

αB-crystallins. These large complexes have chaperone activity and belong to the family of the small heat shock proteins. In contrast to αB-crystallin (gene symbol: Cryab), which is ubiquitously expressed, the αA-crystallin (gene symbol: Cryaa) occurs mainly in the lens. The β/γ-crystallin superfamily exhibits a characteristic protein motif, the so-called Greek key motif, in a quadruple organization. It is considered to be essential for the extremely high protein concentration in the lens (for a review, see Piatigorsky, 2003). Moreover, because of the unique morphology of the lens fiber cells, it is not surprising that certain cytoskeletal proteins are also expressed preferentially in the lens (for a review, see Perng and Quinlan, 2005).

The α-Crystallins and Heat Shock Transcription

Factor When the Cryaa knockout was published (Brady et al., 1997), researchers were surprised that it led to a recessive phenotype and that cataracts became visible only in the homozygous mutants. The opacification starts in the nucleus and progresses to a general opacification with age. Cataract formation ultimately results from insolubility of the αB-crystallin. Cryaa is mapped to mouse chromosome 17; cataract-causing mutations are recessive (Cryaalop18: Chang et al., 1999; Cryaa2J: Xia et al., 2006b) or dominant (CryaaAey7: Graw et al., 2001a; CryaaL1N: Xia et al., 2006a); an example of a dominant phenotype is given in figure 40.2B. The two recessive mutations both affect the Arg54 residue: in the case of the lop18 mutation, it is changed to a His, whereas the Cryaa2J allele is translated at codon 54 into a Cys. In contrast, the dominant mutations affect the C-terminal part of the protein (Aey7: V124E; L1N: Y118D). Similarly, both dominant and recessive CRYAA mutations have been reported in humans: the dominant cataract-causing alleles are R21L, R49C, and R116C (Litt et al., 1998; Mackay et al., 2003; Graw et al., 2006), whereas the nonsense mutation W9X leads to a recessive cataract in an inbred Jewish Persian family (Pras et al., 2000).

Cryab knockout mice (missing αB-crystallin) are cataractfree, but they die prematurely because of myopathy and other organ defects (Brady et al., 2001). Cryab is mapped to mouse chromosome 9; no other Cryab mutation has been reported. In contrast to this finding in the mouse, in humans two different cataract-causing mutations have been described in CRYAB, a dominant myopathy associated with cataract (R120G; Vicart et al., 1998) and a deletion mutation in exon 3 of CRYAB that resulted in a frameshift in codon 150 and an aberrant protein consisting of 184 residues (Berry et al., 2001).

Since the α-crystallins belong to the family of small heat shock proteins, it might be interesting to note that a null mutation in the mouse Hsf4 gene (located on chromosome 8 and coding for heat shock transcription factor 4) causes cataract formation with abnormal lens fiber cells containing

inclusion-like structures (probably due to decreased expression of γ-crystallins; see Fujimoto et al., 2004, and Min et al., 2004). Similarly, a mutation in the human HSF4 gene is associated with a dominant, lamellar cataract (Bu et al., 2002a).

The β-Crystallins The first cataract mutation characterized at a molecular level was the so-called Philly mouse. It was demonstrated to be caused by an in-frame deletion of 12 bp in the βB2-crystallin encoding gene (Crybb2), resulting in a loss of four amino acids (Chambers and Russell, 1991). The region in which the deletion occurs is close to the carboxy-terminus and essential for the formation of the tertiary structure of the βB2-crystallin. The increasing severity of the phenotype is temporally correlated with the expression of the Crybb2 gene; Crybb2 is mapped to mouse chromosome 5. After the first postnatal week, the characteristic bow configuration of the nuclei in the lens cortex was replaced by a fan-shaped configuration, and swelling of the lens fibers occurred. Faint anterior opacities seen at P15 are followed by sutural cataracts at P25, nuclear cataract at P30, lamellar perinuclear opacities at P35, and total nuclear with anterior and posterior polar cataracts at P45. Cataractogenesis is associated with an intralenticular increase in water, sodium, and calcium and a decrease in potassium, reduced glutathione, and ATP. Altered membrane permeability is the cause of an increased outward leak (for a review, see Graw, 2004). A similar phenotype (referred to as Aey2) was also shown to be caused by a mutation affecting the fourth Greek key motif in the βB2-crystallin; however, in this case it was just an amino acid exchange (V187E; Graw et al., 2001b).

Similar to mutations in other crystallin-encoding genes, mutations in the human CRYBB2 gene lead to cataract, too. However, three of the independent human CRYBB2 mutations are caused by gene conversion between CRYBB2 and its closely linked pseudogene, leading to a chain-termination mutation (Vanita et al., 2001). The CRYBB2 pseudogene is specific for the human lineage and does not exist in the mouse genome. A fourth but different CRYBB2 mutation was described recently (W151C in exon 6; Santhiya et al., 2004).

Among the β-crystallin-encoding genes, Cryba1 is the second Cryb gene affected by cataract-causing mutations. It codes for two β-crystallins, βA1and βA3-crystallin, which differ by the length of their N-terminal extension (Peterson and Piatigorsky, 1986). In the mouse, one Cryba1 mutation has been described (progressive opacity, Po1), which has a similar phenotype to the murine Crybb2 mutations cited earlier. It is characterized by a splicing defect at the end of intron 5, which leads to two distinct mRNA products and therefore to different predicted proteins: the Trp at position 168 is either deleted or changed into an Arg (Graw et al., 1999).

Similarly, in humans, two independent mutations affect the same 5′ (donor) splice site of intron 3. However, there is usually no access to human lens cDNA, and the novel splice product could not be characterized (for a review, see Graw, 2003). The third mutation was reported as a G91 deletion, causing a lamellar cataract with variable severity (Reddy et al., 2004). In humans, additional cataract-causing mutations in CRYB genes have been described in CRYBB1 (Mackay et al., 2002) and CRYBA4 (Billingsley, 2006); however, in the mouse, the other Cryb genes are not yet reported to be targeted by a mutation.

The γ-Crystallins An intermediate member of the β/γ- crystallin superfamily is γS-crystallin, previously also referred to as βS-crystallin. The corresponding gene Crygs maps to mouse chromosome 16, and two dominant mutations have been shown to be associated with Crygs: the ENU-induced, dominant mutation Opj (opacity due to poor junctions) was shown to have a mutation coding for a key residue in the core of the N-terminal domain of the protein (Phe9Ser; Sinha et al., 2001). In contrast, the spontaneous recessive mutation in mouse Crygs is characterized by a stop codon leading to a truncated protein missing 16 amino acids at the C-terminus of the mouse Crygs gene (Bu et al., 2002b).

The other six Cryg genes are organized as a cluster of very similar genes (Cryga→Crygf) within approximately 50 kb on mouse chromosome 1. In mice, mutations have been characterized affecting all six genes; however, it is apparent that Cryge has the highest mutation frequency. An overview of about 20 characterized mouse mutants has been published recently (Graw et al., 2004). Two very different phenotypes are presented in figure 40.3. The first Cryg mutant to be identified was the Elo mutant (eye lens obsolescence); it was characterized by a single nucleotide deletion in the Cryge gene. The mutation destroys the reading frame of the gene, and at the protein level one of the Greek key motifs is affected (Cartier et al., 1992). One of the cataract mutants most characterized among this group was originally referred to as Nop (nuclear opacity). It was of spontaneous origin and shown to be caused by a small deletion of 11 bp and an insertion of 4 bp in the third exon of the Crygb gene (allele symbol: Crygbnop). It leads to a frameshift and ultimately creates a new stop codon; the corresponding γB-crystallin protein is predicted to be truncated after 144 amino acids; the last six amino acids are different from the wild-type γBcrystallin. Western blot analysis demonstrated stable expression of the wrong protein (Klopp et al., 1998). An additional, ENU-induced Crygb mutation (I4F) was described recently by Liu et al. (2005). The authors discuss a higher affinity to α-crystallin as the major cause of cataract formation in this particular mutant.

All these Cryg mutations affect only the lens cells and no other part of the eye; however, the size of the entire eye is