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Generating mouse models of retinal disease using ENU mutagenesis
Vision Research 42 (2002) 479–485 www.elsevier.com/locate/visres
Generating mouse models of retinal disease using ENU mutagenesis P.N. Baird a,*, R.H. Guymer a, D. Chiu a, A.L. Vincent a,b, W.S. Alexander c, S.J. Foote c, D.J. Hilton c a
University of Melbourne, Centre for Eye Research Australia (CERA), University of Melbourne, 32 Gisborne Street, East Melbourne, Victoria 3002, Australia b Vision Science Research Program, University Health Network, Toronto, Canada c The Walter and Eliza Hall, Institute of Medical Research, Melbourne, Australia Received 4 May 2001; received in revised form 7 August 2001
Abstract We used the chemical mutagen, N-ethyl-N-nitrosourea, to induce random point mutations in the germline of the mouse strain C57BL/6 in order to generate models of retinal diseases. 1163 mutagenised first generation mice produced using this approach were examined for eye abnormalities. Approximately one-third (412) presented with some form of ocular abnormality. Most changes were unilateral and confined to the anterior segment of the eye. Less than 10% (44) of identified changes affected the posterior segment of the eye. 21 mice with varying ocular abnormalities, including 17 with retinal changes, were bred to produce second generation mice to confirm genetic inheritance. Genetic inheritance was confirmed in several of these lines including three with retinal changes. Ó 2002 Elsevier Science Ltd. All rights reserved. Keywords: Disease; Genetics; Mouse; Mutant; Retina
1. Introduction Abnormalities of the retina represent a phenotypically heterogeneous group of diseases. Diabetic retinopathy and age-related macular degeneration (AMD) account for the majority of vision impairment and blindness within this group of diseases. Both of these diseases appear to be complex genetic disorders with environmental risk factors impacting on genetic background, (Heiba, Elston, Klein, & Klein, 1994). Little is known about the genetic causes of either disease, although both are increasing in prevalence. To unravel some of the genetic events behind these diseases we have sought to develop an animal model of retinal disease. The mouse has proven particularly useful due to the ease of genetic manipulation, its rapid reproduction rate and its similarity in genomic structure to humans. A number of mutant mouse models of retinal disease are available through either spontaneous muta*
Corresponding author. Tel.: +61-3-9929-8613; fax: +61-3-96623859. E-mail address: [email protected] (P.N. Baird).
tions, including the rds (van Nie, Ivanyi, & Demant, 1978) and tubby mouse, (Ikeda et al., 2000) or through the use of induced mutations that may arise from transgenic or targeted alteration. However there is still a fundamental gap between the number of identified loci involved in retinal disease and the number of appropriate mouse models. Our strategy to dissect out the genetic events in retinal disease was via the use of mutagenesis using the chemical agent N-ethyl-N-nitrosourea (ENU). This mutagen has been used to great effect in several organisms including the mouse, (Russell et al., 1982) and has led to the isolation of the ‘‘clock’’ gene involved in circadian rhythm (Vitaterna et al., 1994) and in identifying the Cyb1 gene involved in cataract formation (Graw et al., 1999). We used a mouse breeding strategy to generate dominant mutations where an altered phenotype could be readily detected. The use of ENU mutagenesis requires no prior knowledge about the underlying genetic events involved in disease and thus provides a powerful means of identifying novel genes and their pathways in disease. The inbred mouse strain C57BL/6 was used in this study, as previous reports have only reported the
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P.N. Baird et al. / Vision Research 42 (2002) 479–485
occurrence of spontaneous congenital ocular abnormalities affecting the anterior segment of the eye. These anterior abnormalities consist of microphthalmia, anophthalmia and cataracts (Cook & Sulik, 1986; Robinson, Holmgren, & Dewey, 1993). The first generation (G1) progeny of ENU-treated male mice were screened for ocular phenotypes. Ophthalmic examination identified a number of mouse lines of interest. Several of these lines resulted in retinal changes and were followed for disease progression over a time course of 18 months to two years. Breeding was used to confirm that a phenotype of interest resulted from a genetically inherited mutation.
methoxyflurane (Medical Developments Australia Pty. Ltd., Victoria). Following anesthesia, the pupil of each eye was dilated using a single drop of 0.5% Tropicamide (Alcon Labs., NSW). Anterior and posterior segments of both eyes were ophthalmalogically examined using an operating microscope (Scanoptics, model SO-111, Adelaide, SA). The lids and anterior segments were examined directly under the microscope. Posterior segment visualization was achieved using a 10 mm round coverslip, with a drop of saline placed on the cornea. Ethical approval for this procedure was through the Royal Melbourne Hospital Research Foundation Animal Ethics Committee (AEC 1999/007). 2.4. Ocular photography
2. Methods 2.1. Animal husbandry All experiments were performed in compliance with the National Health and Medical Research Council of Australia guidelines on animal experimentation. C57BL/ 6 mice were housed at no more than eight animals in a cage in the presence of white pine bedding. The environment was kept at a constant temperature of 22 2 °C in light/dark cycles, each of 12 h. 2.2. Generation of mutant mice 80 eight-week-old male C57BL/6 mice were raised in special pathogen-free (SPF) facilities. Half the mice were injected intra-peritoneal with a single dose of 150 mg/kg that was estimated to induce mutations at the rate of 150 105 or a locus specific rate of approximately 1 in 700 male gametes (Hitotsumachi, Carpenter, & Russell, 1985). The remaining mice were injected with three doses of 100 mg/kg ENU as previously described (Bode, 1984). After one month, two animals from each group were sacrificed and their testes examined histologically and showed the clear effects of ENU injection. Following ENU injection there was a short sterile period of between 9 and 20 weeks after which G1 offspring were generated. 34 animals from the group injected with a single dose of ENU and 11 mice injected with three doses of ENU were mated with normal (non-mutagenised) C57BL/6 females to produce 1163 G1 animals. The other mice either became sick and were sacrificed prior to producing offspring or had not regained their fertility 9 months after ENU injection.
A hand held Kowa Genesis human fundus camera was used to photograph both anterior and posterior segment changes as previously described (Hawes et al., 1999). Fundi were examined using the camera in association with a hand held lens of either 66 diopter (D) or 90D. 64 or 100ASA slide film (Kodak) was used to document changes.
3. Results 3.1. Mice examined In this study, the mutagen, ENU, was used to introduce random point mutations into the germline of male C57BL/6 mice. Mating of these males to wild type C57BL/6 female mice resulted in the production of 1163 G1 offspring available for screening in this study (Fig.
2.3. Ophthalmic examination 1163 G1 mice were available for ophthalmic analysis. Mice were transferred from the SPF facility to a conventional clean animal facility and were anesthetized (approximately 5 min) in a covered jar using 1ml of
Fig. 1. ENU mutagenesis strategy used to generate mice with ocular phenotypes.
P.N. Baird et al. / Vision Research 42 (2002) 479–485
3.3. Anterior segment changes
Table 1 Number of mice and eye abnormalities Number of mice Number with ocular abnormality
481
Male (%)
Female (%)
Total
608 (52) 166 (27)
555 (48) 204 (37)
1163 370
1). Initial ophthalmic examination of G1 mice was at a minimum of 8 weeks, but the majority of examinations were undertaken between 15–20 weeks and all were conducted within a period of 6 months. Several mice with ocular abnormalities were examined over a period of up to eighteen months to document any progression in phenotypic presentation.
Abnormality by disease type allowed six groups to be assigned for anterior changes. These groups were fleck cataracts, tumescent cataract, corneal opacity with or without cataract, all with normal size eyes, anterior segment disruption including cataract, microphthalmia (including anophthalmia) and lid abnormalities (Table 2). Most of the increased number of abnormalities in female mice arose from the fleck cataracts, corneal opacity with or without cataract and microphthalmia groups (Table 2). In contrast however, tumescent cataract exhibited a preponderance of changes in the male 14 (64%) compared to 8 (36%) in female mice (Table 2).
3.2. Ocular changes by sex
3.4. Posterior segment changes
Approximately equal numbers of 555 female (48%) and 608 (52%) male mice offspring (Table 1) were ophthalmologically examined. An ocular abnormality was detected in a total of 370 (32%) of these G1 mice. Of the male mice 27% (166/608) presented with an ocular abnormality compared to 37% (204/555) for female mice, indicating a slight sex bias for phenotypic abnormalities in favor of females (Table 1). Phenotypic changes detected were first subdivided by either anterior or posterior segment in either male or female mice (Table 2) and then grouped by ocular abnormality (Table 2). Of the 370 ocular abnormalities detected, the majority, 326 (88%) occurred in the anterior segment of the eye and only 44 (12%) in the posterior segment (Table 2). In both anterior and posterior abnormalities there was a sex bias towards female mice presenting with abnormalities. In the case of anterior changes this was 180 (55%) compared to 146 (45%) and in posterior changes, it was 24 (55%) compared to 20 (45%) in female and male mice respectively (Table 2).
In posterior changes, the abnormalities detected were assigned to one of two groups being either disc changes or retinal changes. There appeared to be an excess of female cases with disc changes although the numbers were too small to draw any conclusions (Table 2). Mice with retinal abnormalities however showed an equal distribution between the sexes with 19 (49%) abnormalities occurring in female mice and 20 (51%) in male mice (Table 2). Of the posterior changes, all presented as either retinal detachment, areas of hypo- or hyper-pigmentation, whitish plaques or spots, formation of aberrant blood vessels or retinal hemorrhage.
Table 2 Eye abnormalities detected in G1 offspring based on sex, location and disease type Group
Type of abnormality
Male
Female
Total
Anterior
Tumescent cataract/ normal size Segment disruption with cataract Microphthalmic Corneal opacity cataract Fleck cataracts Lid abnormality Subtotal
14
8
22
10
11
21
29 26
40 34
69 60
62 5 146
83 4 180
145 9 326
1 19 20
4 20 24
5 39 44
166
204
370
Posterior
Pole––discs Pole––retina Subtotal Total
3.5. Ocular changes by eye When ocular abnormalities were analyzed depending on the eye of presentation, most changes were unilateral. Of the 326 anterior changes, 286 (88%) were unilateral with only 40 (12%) being bilateral (Table 3). In posterior cases, this figure was 39/44 (89%) for unilateral and 5/44 (11%) for bilateral (Table 3). Abnormalities within each eye were not evenly distributed in either anterior or posterior cases. In anterior defects, the right eye presented with 213/326 (65%) and the left eye 73/326 (22%) (not including bilaterals) indicating a severe bias to the right eye in terms of phenotypic change. In posterior cases, 29/40 (72.5%) cases presented in the right eye compared to 10/40 (25%) in the left eye (Table 3). Although the majority of mouse eye phenotypes observed in this screen were anterior in presentation, we were particularly interested in posterior changes that might resemble human retinal diseases. We did not detect any phenotypes that resembled retinitis pigmentosa, nor diabetic retinopathy but did identify some phenotypes that resembled AMD. In the case of mouse line 154.6, a number of white lesions were present that appeared to mimic the drusen seen in AMD and in mouse line 131.27, regions of hypo- and hyper-pigmentation
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Table 3 Abnormalities detected in G1 offspring by eye Group
Type of abnormality
Right
Left
Anterior
Tumescent cataract/ normal size Segment disruption with cataract Microphthalmic Corneal opacity cataract Fleck Cataracts Lid abnormality Subtotal
19
1
2
17
4
0
60 45
6 7
3 8
64 8 213
54 1 73
27 0 40
5 24 29
0 10 10
0 5 5
242
83
45
Posterior
Pole––discs Pole––retina Subtotal Total
Bilateral
were present. Mouse fundi photographs were taken to demonstrate the changes (Fig. 2). 3.6. Mouse lines for breeding A total of 30 G1 mice with different phenotypes of interest were initially selected for breeding in this study to confirm genetic inheritance. These consisted of 25 retinal and five anterior phenotypes. Two mouse lines however did not achieve mating (one posterior and one anterior) due to the old age of the mice. One posterior mouse line died giving birth. Eight of the posterior lines did not produce offspring and breeding was therefore attempted using an assisted reproductive technique of either ‘in vitro fertilization’ (IVF), ‘egg transplantation’, or ‘ovarian transplantation’. In general, IVF was successful (two out of three mice) but egg transplantation and ovarian transplantation ovarian were unsuccessful. This left a total of 17 mice with retinal phenotypes and four mice with anterior phenotypes from which to breed. 3.7. Confirmation of genetic inheritance It was necessary to confirm that the phenotypic abnormality detected was due to an inherited genetic change. As the breeding strategy targeted dominantly induced ENU mutations, a maximum of 50% of offspring should have inherited the causative mutation. Of the 17 retinal phenotypes produced (Table 4), an average of 13 second generation (G2) offspring were produced for each line. With regard to the anterior defects, an average of 8 G2 offspring per line were produced (Table 4). Genetic inheritance was confirmed in both posterior and anterior lines. Two of the four anterior lines produced offspring with the same phenotype as the parent (Table 4). Inheritance in the anterior mouse line 46.15 was 43% (3/7 G2 mice) and in mouse line 19.2, this was
50% (2/4 G2 mice) (Table 4). In the case of the 17 posterior lines, only 3 of the lines appeared to confer genetic inheritance (Table 4). In the case of mouse line 39.7, only 1/12 G2 mice (8%) had an inherited phenotype (Table 4). In mouse line 129.10, this was 2/13 G2 mice (15%) (Table 4) and in mouse line 131.27, 5/14 G2 mice (36%) had an inherited phenotype (Table 4). Of the mice that presented with a similar phenotype to the parent, there did not appear to be any bias in terms of the location within a particular eye (Table 4). In terms of the sex of the G2 offspring, both male and females were represented in the anterior abnormalities (Table 4). In posterior cases, lines 39.7 and 129.10, G2 offspring were only found in female mice although the numbers were small at 1 and 2 mice respectively (Table 4). In the case of mouse line 131.27, all five G2 offspring were in male mice (Table 4).
4. Discussion It is known that a number of retinal phenotypes result from mutational events in single genes, we were interested however in exploring the use of ENU mutagenesis to dissect out components of complex diseases such as diabetic retinopathy and AMD. Our strategy was therefore based on dissecting out single gene mutational events, which on the C57BL/6 genetic background have pleiotrophic effects relevant to retinal disease phenotypes. These mutational events can give rise to either hypomorphic changes (partial loss of function), gain of function or total loss of function (Justice & Bode, 1988). Although some of these mutational events may be rare, complex diseases result as a consequence of many different genes and therefore the probability of detecting one of these genes is increased. In order to try to identify later occurring retinal abnormalities in mice, we chose to perform retinal exams at an age of between 15–20 weeks. It was anticipated that examining mice retina at this age would allow us to detect a broad range of phenotypes whilst still allowing breeding for further genetic studies. Our confidence in this approach was boosted by the identification of a number of mice that presented with retinal phenotypes of interest. All mice with retinal abnormalities were bred to confirm genetic inheritance. The three G1 mouse lines that showed genetic inheritance resulting in posterior changes were followed for disease progression over a period of 18 months. The original eye exam of the G1 mouse line 131.27, defined regions of hypo- and hyperpigmented retinal areas of the right eye and this finding did not change over the next 18 months (Fig. 2). In addition, the other retinal lines examined over this 18month time period did also not appear to progress from the original description given in the eye examination at 15–20 weeks.
P.N. Baird et al. / Vision Research 42 (2002) 479–485
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Fig. 2. Fundus photographs of posterior segment abnormalities: (a) Mouse line 131.27 showing a large white and several smaller whitish areas of retinal hypo-pigmentation and (b) Mouse line 154.6 showing multiple white retinal lesions.
In this study we detected a large number of ocular abnormalities. These included 326 with anterior segment changes and 44 with posterior segment changes of the eye. The predominance of anterior changes agrees with previously published reports on C57BL/6 inbred mice. These changes consisted mainly of microphthalmia, anophthalmia and cataracts (Cook & Sulik, 1986; Robinson et al., 1993); and a low frequency of corneal opacity (Harch, Chase, & Gonsalves, 1978; Cook & Sulik, 1986). Previous reports however have indicated that spontaneous congenital abnormalities occur at between 5% and 15% (Cook & Sulik, 1986; Robinson et al., 1993); whilst in our study these occurred at 28% (326/ 1163). This approximate doubling of the previously reported rate suggests that ENU mutagenesis had led to
this increase in abnormalities. Although we noted a high rate of background phenocopies, our genetic analysis did show inheritance in two lines with cataract/corneal opacity. This again suggested that the introduction of a germline mutation in G0 founder mice had been inherited through G1 and G2 mice (Table 4). The appearance of posterior segment abnormalities, previously not reported, but found in this study, adds to our belief that ENU resulted in mutagenesis. Overall in our study, both anterior and posterior segment abnormalities were 1.23 times more frequent in females compared to male mice. In the case of anterior segment changes, these presented as 180 in females compared to 146 in males and for posterior segment changes, these were 204 in females compared to 166 in
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Table 4 Inheritance of eye abnormality Mouse ID
Sex
Eye
Abnormality
Offspring inheriting abnormality
% inherited
Sex of inherited mouse
Eye of inherited mouse
Anterior 46.15
Female
Right
3/7
43
Female/Male
Left/Right
66.8
Female
Right
0/9
0
31.28 19.2
Female Female
Bilat Bilat
Corneal peripheral haze/post polar cat Microphthalmia/lower lid granuloma Hazy cornea Corneal opacity/ant polar cataract
0/12 2/4
0 50
Female/Male
Left/Right
Posterior 32.11 39.7 147.1 61.8 129.10 13.11 127.8 28.9
Male Male Female Male Male Female Female Female
Left Right Right Right Left Right Left Left
0/13 1/12 0/12 0/14 2/13 0/7 0/9 0/9
0 8 0 0 15 0 0 0
Female
Right
Female
Left/Right
132.4 143.10 168.15
Male Female Female
Right Right Right
145.26 131.27
Female Male
Right Right
Male
Left/Right
19.61 32.56
Female Male
Right Right
154.6 66.13
Female Male
Right Left
Superonasal hypo-pigmentation Disc Cupped disc Nasal hyper-pigmentation Multiple white lesions Raised retina White plaque/subretinal lesion Hypo-pigmented supranasal region Subretinal area of whiteness Pale large disc Hypo- & hyper-pigmented retinal areas Subretinal area of whiteness Hypo- & hyper-pigmented retinal areas Retinal detachment Mottled pigmentary retinal changes White peripheral retinal dots Hypo-pigmented dots
males (Table 2). The only exceptions to this were in the cases of tumescent cataracts and also in lid abnormalities where there was a reversal in the sex bias (Table 2). This alteration of the sex bias compared to previous studies may reflect either a difference in environmental factors or more likely it reflects the use of ENU. We know from our data that ENU does not affect the sex of offspring, as approximately equal numbers of both male and female mice were produced in the study (Table 1). It would therefore imply that either more male mice inherit ENU induced mutations, giving rise to abnormalities, or that environmental factors present in this study resulted in less female abnormalities. This situation could be better clarified if a repeat study was performed on nonENU treated C57BL/6 mice under the same conditions as above. The number of bilateral G1 mice produced as a result of ENU mutagenesis was small in anterior cases and minimal in posterior cases. Anterior bilateral abnormalities were approximately 7.15 times less frequent than unilateral changes (40/213þ73) (Table 3). In posterior cases however, only one of the five G1 mouse lines identified with bilateral retinal changes had a phenotype
In progress 0/5 0/15
– 0 0
0/13 5/14
0 36
0/14 0/13
0 0
0/11 0/15
0 0
of interest but unfortunately died giving birth. The reason for this unilateral bias is unknown but our breeding studies have indicated that mice with unilateral changes do show genetic inheritance (Table 4). Previous reports have also indicated that right eye anterior segment abnormalities are up to 5.8 times more common than left eye abnormalities in C57BL/6 mice (Pierro & Spiggle, 1967). In this study we also found a bias between eyes although only at half the rate previously reported (2.9 times) (Table 3). Genetic inheritance in this study appeared variable with 2/4 anterior segment mouse lines and 3/17 posterior segment mouse lines exhibiting genetic inheritance (Table 4). It was noted that the level of penetrance observed in the mouse lines was variable. In the two anterior lines, penetrance was close to 50% (Table 4) as may be expected for a dominant mutation. In the case of the three posterior mouse lines however the penetrance varied from a maximum of 36% in mouse line 131.27 down to 8% in mouse line 39.7 (Table 4). Most of the 17 G1 mouse lines that presented with retinal changes resembled the early stages of AMD. In the case of mouse line 154.6 multiple white lesions were present that
P.N. Baird et al. / Vision Research 42 (2002) 479–485
appeared to mimic the drusen deposits found in early AMD (Fig. 2). In the case of retinal line 131.27, regions of hypo- and hyper-pigmented retinal areas were seen (Fig. 2), again characteristic of early stages of AMD. The variability in penetrance observed in the 17 G2 retinal lines may reflect the presence of a hypomorphic change where there is only a partial loss of function (Justice & Bode, 1988). It may also reflect the development of a phenotype that occurs later in the mouse lifespan and therefore these mice need to be followed and examined over a longer time frame. This is currently taking place. Further breeding is also underway to produce more G2 offspring from these mouse lines of interest to allow both a better definition of penetrance and also to allow for histological examination. If changes similar to AMD are confirmed following histological examination then further breeding will be conducted to allow genetic linkage studies to be performed and allow identification of the causative gene. The use of ENU clearly provides the ability to generate mouse models of human disease. We have demonstrated that the use of this mutagen is capable of producing mouse retinal phenotypes that resemble human disease. The experimental procedures however can be complicated by the number of spontaneous phenotypic changes that can occur (in the case of anterior segment changes) and the variable penetrance. A welldefined breeding strategy to confirm genetic inheritance is therefore necessary to confirm the penetrance and nature of a causative mutation. We believe that the use of this technique may be of merit in generating new models of mouse retinal disease and hope to extend our findings through the elucidation of equivalent genes involved in human retinal disease.
Acknowledgements We thank all the animal house technicians who took part in the maintenance of animals both at Kew and Parkville campuses of WEHI. This work was supported by a grant from the Ophthalmic Research Institute of Australia and an NHMRC block grant to the Walter and Eliza Hall Institute of Medical Research.
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References Bode, V. C. (1984). Ethylnitrosourea mutagenesis and the isolation of mutant alleles for specific genes located in the T region of mouse chromosome 17. Genetics, 108, 457–470. Cook, C. S., & Sulik, K. K. (1986). Sequential scanning electron microscopic analyses of normal and spontaneously occurring abnormal ocular development in C57BL/6J mice. Scanning Electron Microscopy, 111, 1215–1227. Graw, J., Jung, M., Loster, J., Klopp, N., Soewarto, D., Fella, C., Fuchs, H., Reis, A., Wolf, E., Balling, R., & Hrabe de Angelis, M. (1999). Mutation in the bA3/A1-Crystallin encoding gene Cryba1 causes dominant cataract in the mouse. Genomics, 62, 67–73. Harch, C., Chase, H. B., & Gonsalves, N. I. (1978). Studies on an anophthalmic strain of mice. Vl. Lens and cup interaction. Developmental Biology, 124, 352–357. Hawes, N. L., Smith, R. S., Chang, B., Davisson, M., Heckenlively, J. R., & John, S. W. M. (1999). Mouse fundus photography and angiography: A catalogue of normal and mutant phenotypes. Molecular Vision, 5, 22–29. Heiba, I. M., Elston, R. C., Klein, B. E., & Klein, R. (1994). Sibling correlations and segregation analysis of age-related maculopathy: the Beaver Dam Eye Study. Genetic Epidemiology, 11, 51–67. Hitotsumachi, S., Carpenter, D. A., & Russell, W. L. (1985). Doserepetition increases the mutageneic effectiveness of N-ethylN-nitrosourea in mouse spermatogonia. Proceedings National Academy Science, USA, 82, 6619–6621. Ikeda, S., Shival, N., Ikeda, A., Smith, R. S., Nusinowitz, S., Yan, G., Lin, T. R., Chu, S., Heckenlively, J. R., North, M. A., Naggert, J. K., Nishina, P. M., & Duyao, M. P. (2000). Retinal degeneration but not obesity is observed in null mutants of the tubby-like protein1 gene. Human Molecular Genetics, 9, 155–163. Justice, M. J., & Bode, V. (1988). Genetic analysis of mouse t haplotypes using mutations induced by ethylnitrosurea muagenesis: the order of T and qk is inverted in t mutants. Genetics, 120, 533–543. Pierro, L. J., & Spiggle, J. (1967). Congenital eye defects in the mouse. l. Corneal opacity in C57 Black mice. Journal of Experimental Zoology, 166, 25–38. Robinson, M. L., Holmgren, A., & Dewey, M. J. (1993). Genetic control of ocular morphogenesis: Defective lens development associated with ocular anomalies in C57BL/6 mice. Experimental Eye Research, 56, 7–16. Russell, W. L., Hunsicker, P. R., Raymer, G. D., Steele, M. H., Stelzner, K. F., & Thompson, H. M. (1982). Dose-response curve for ethylnitrosourea-induced specific-locus mutations in mouse spermatogonia. Proceedings National Academy Science, USA, 79, 3589–3591. van Nie, R., Ivanyi, D., & Demant, P. (1978). A new H-2-linked mutation, rds, causing retinal degeneration in the mouse. Tissue Antigen, 12, 106–108. Vitaterna, M. H., King, D. P., Chang, A. M., Kornhauser, J. M., Lowrey, P. L., McDonald, J. D., Dove, W. F., Pinto, L. H., Turek, F. W., & Takahashi, J. S. (1994). Mutagenesis and mapping of a mouse gene, Clock, essential for circadian behavior. Science, 264, 719–725.
Generating mouse models of retinal disease using ENU mutagenesis P.N. Baird a,*, R.H. Guymer a, D. Chiu a, A.L. Vincent a,b, W.S. Alexander c, S.J. Foote c, D.J. Hilton c a
University of Melbourne, Centre for Eye Research Australia (CERA), University of Melbourne, 32 Gisborne Street, East Melbourne, Victoria 3002, Australia b Vision Science Research Program, University Health Network, Toronto, Canada c The Walter and Eliza Hall, Institute of Medical Research, Melbourne, Australia Received 4 May 2001; received in revised form 7 August 2001
Abstract We used the chemical mutagen, N-ethyl-N-nitrosourea, to induce random point mutations in the germline of the mouse strain C57BL/6 in order to generate models of retinal diseases. 1163 mutagenised first generation mice produced using this approach were examined for eye abnormalities. Approximately one-third (412) presented with some form of ocular abnormality. Most changes were unilateral and confined to the anterior segment of the eye. Less than 10% (44) of identified changes affected the posterior segment of the eye. 21 mice with varying ocular abnormalities, including 17 with retinal changes, were bred to produce second generation mice to confirm genetic inheritance. Genetic inheritance was confirmed in several of these lines including three with retinal changes. Ó 2002 Elsevier Science Ltd. All rights reserved. Keywords: Disease; Genetics; Mouse; Mutant; Retina
1. Introduction Abnormalities of the retina represent a phenotypically heterogeneous group of diseases. Diabetic retinopathy and age-related macular degeneration (AMD) account for the majority of vision impairment and blindness within this group of diseases. Both of these diseases appear to be complex genetic disorders with environmental risk factors impacting on genetic background, (Heiba, Elston, Klein, & Klein, 1994). Little is known about the genetic causes of either disease, although both are increasing in prevalence. To unravel some of the genetic events behind these diseases we have sought to develop an animal model of retinal disease. The mouse has proven particularly useful due to the ease of genetic manipulation, its rapid reproduction rate and its similarity in genomic structure to humans. A number of mutant mouse models of retinal disease are available through either spontaneous muta*
Corresponding author. Tel.: +61-3-9929-8613; fax: +61-3-96623859. E-mail address: [email protected] (P.N. Baird).
tions, including the rds (van Nie, Ivanyi, & Demant, 1978) and tubby mouse, (Ikeda et al., 2000) or through the use of induced mutations that may arise from transgenic or targeted alteration. However there is still a fundamental gap between the number of identified loci involved in retinal disease and the number of appropriate mouse models. Our strategy to dissect out the genetic events in retinal disease was via the use of mutagenesis using the chemical agent N-ethyl-N-nitrosourea (ENU). This mutagen has been used to great effect in several organisms including the mouse, (Russell et al., 1982) and has led to the isolation of the ‘‘clock’’ gene involved in circadian rhythm (Vitaterna et al., 1994) and in identifying the Cyb1 gene involved in cataract formation (Graw et al., 1999). We used a mouse breeding strategy to generate dominant mutations where an altered phenotype could be readily detected. The use of ENU mutagenesis requires no prior knowledge about the underlying genetic events involved in disease and thus provides a powerful means of identifying novel genes and their pathways in disease. The inbred mouse strain C57BL/6 was used in this study, as previous reports have only reported the
0042-6989/02/$ - see front matter Ó 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 0 4 2 - 6 9 8 9 ( 0 1 ) 0 0 2 3 2 - 2
480
P.N. Baird et al. / Vision Research 42 (2002) 479–485
occurrence of spontaneous congenital ocular abnormalities affecting the anterior segment of the eye. These anterior abnormalities consist of microphthalmia, anophthalmia and cataracts (Cook & Sulik, 1986; Robinson, Holmgren, & Dewey, 1993). The first generation (G1) progeny of ENU-treated male mice were screened for ocular phenotypes. Ophthalmic examination identified a number of mouse lines of interest. Several of these lines resulted in retinal changes and were followed for disease progression over a time course of 18 months to two years. Breeding was used to confirm that a phenotype of interest resulted from a genetically inherited mutation.
methoxyflurane (Medical Developments Australia Pty. Ltd., Victoria). Following anesthesia, the pupil of each eye was dilated using a single drop of 0.5% Tropicamide (Alcon Labs., NSW). Anterior and posterior segments of both eyes were ophthalmalogically examined using an operating microscope (Scanoptics, model SO-111, Adelaide, SA). The lids and anterior segments were examined directly under the microscope. Posterior segment visualization was achieved using a 10 mm round coverslip, with a drop of saline placed on the cornea. Ethical approval for this procedure was through the Royal Melbourne Hospital Research Foundation Animal Ethics Committee (AEC 1999/007). 2.4. Ocular photography
2. Methods 2.1. Animal husbandry All experiments were performed in compliance with the National Health and Medical Research Council of Australia guidelines on animal experimentation. C57BL/ 6 mice were housed at no more than eight animals in a cage in the presence of white pine bedding. The environment was kept at a constant temperature of 22 2 °C in light/dark cycles, each of 12 h. 2.2. Generation of mutant mice 80 eight-week-old male C57BL/6 mice were raised in special pathogen-free (SPF) facilities. Half the mice were injected intra-peritoneal with a single dose of 150 mg/kg that was estimated to induce mutations at the rate of 150 105 or a locus specific rate of approximately 1 in 700 male gametes (Hitotsumachi, Carpenter, & Russell, 1985). The remaining mice were injected with three doses of 100 mg/kg ENU as previously described (Bode, 1984). After one month, two animals from each group were sacrificed and their testes examined histologically and showed the clear effects of ENU injection. Following ENU injection there was a short sterile period of between 9 and 20 weeks after which G1 offspring were generated. 34 animals from the group injected with a single dose of ENU and 11 mice injected with three doses of ENU were mated with normal (non-mutagenised) C57BL/6 females to produce 1163 G1 animals. The other mice either became sick and were sacrificed prior to producing offspring or had not regained their fertility 9 months after ENU injection.
A hand held Kowa Genesis human fundus camera was used to photograph both anterior and posterior segment changes as previously described (Hawes et al., 1999). Fundi were examined using the camera in association with a hand held lens of either 66 diopter (D) or 90D. 64 or 100ASA slide film (Kodak) was used to document changes.
3. Results 3.1. Mice examined In this study, the mutagen, ENU, was used to introduce random point mutations into the germline of male C57BL/6 mice. Mating of these males to wild type C57BL/6 female mice resulted in the production of 1163 G1 offspring available for screening in this study (Fig.
2.3. Ophthalmic examination 1163 G1 mice were available for ophthalmic analysis. Mice were transferred from the SPF facility to a conventional clean animal facility and were anesthetized (approximately 5 min) in a covered jar using 1ml of
Fig. 1. ENU mutagenesis strategy used to generate mice with ocular phenotypes.
P.N. Baird et al. / Vision Research 42 (2002) 479–485
3.3. Anterior segment changes
Table 1 Number of mice and eye abnormalities Number of mice Number with ocular abnormality
481
Male (%)
Female (%)
Total
608 (52) 166 (27)
555 (48) 204 (37)
1163 370
1). Initial ophthalmic examination of G1 mice was at a minimum of 8 weeks, but the majority of examinations were undertaken between 15–20 weeks and all were conducted within a period of 6 months. Several mice with ocular abnormalities were examined over a period of up to eighteen months to document any progression in phenotypic presentation.
Abnormality by disease type allowed six groups to be assigned for anterior changes. These groups were fleck cataracts, tumescent cataract, corneal opacity with or without cataract, all with normal size eyes, anterior segment disruption including cataract, microphthalmia (including anophthalmia) and lid abnormalities (Table 2). Most of the increased number of abnormalities in female mice arose from the fleck cataracts, corneal opacity with or without cataract and microphthalmia groups (Table 2). In contrast however, tumescent cataract exhibited a preponderance of changes in the male 14 (64%) compared to 8 (36%) in female mice (Table 2).
3.2. Ocular changes by sex
3.4. Posterior segment changes
Approximately equal numbers of 555 female (48%) and 608 (52%) male mice offspring (Table 1) were ophthalmologically examined. An ocular abnormality was detected in a total of 370 (32%) of these G1 mice. Of the male mice 27% (166/608) presented with an ocular abnormality compared to 37% (204/555) for female mice, indicating a slight sex bias for phenotypic abnormalities in favor of females (Table 1). Phenotypic changes detected were first subdivided by either anterior or posterior segment in either male or female mice (Table 2) and then grouped by ocular abnormality (Table 2). Of the 370 ocular abnormalities detected, the majority, 326 (88%) occurred in the anterior segment of the eye and only 44 (12%) in the posterior segment (Table 2). In both anterior and posterior abnormalities there was a sex bias towards female mice presenting with abnormalities. In the case of anterior changes this was 180 (55%) compared to 146 (45%) and in posterior changes, it was 24 (55%) compared to 20 (45%) in female and male mice respectively (Table 2).
In posterior changes, the abnormalities detected were assigned to one of two groups being either disc changes or retinal changes. There appeared to be an excess of female cases with disc changes although the numbers were too small to draw any conclusions (Table 2). Mice with retinal abnormalities however showed an equal distribution between the sexes with 19 (49%) abnormalities occurring in female mice and 20 (51%) in male mice (Table 2). Of the posterior changes, all presented as either retinal detachment, areas of hypo- or hyper-pigmentation, whitish plaques or spots, formation of aberrant blood vessels or retinal hemorrhage.
Table 2 Eye abnormalities detected in G1 offspring based on sex, location and disease type Group
Type of abnormality
Male
Female
Total
Anterior
Tumescent cataract/ normal size Segment disruption with cataract Microphthalmic Corneal opacity cataract Fleck cataracts Lid abnormality Subtotal
14
8
22
10
11
21
29 26
40 34
69 60
62 5 146
83 4 180
145 9 326
1 19 20
4 20 24
5 39 44
166
204
370
Posterior
Pole––discs Pole––retina Subtotal Total
3.5. Ocular changes by eye When ocular abnormalities were analyzed depending on the eye of presentation, most changes were unilateral. Of the 326 anterior changes, 286 (88%) were unilateral with only 40 (12%) being bilateral (Table 3). In posterior cases, this figure was 39/44 (89%) for unilateral and 5/44 (11%) for bilateral (Table 3). Abnormalities within each eye were not evenly distributed in either anterior or posterior cases. In anterior defects, the right eye presented with 213/326 (65%) and the left eye 73/326 (22%) (not including bilaterals) indicating a severe bias to the right eye in terms of phenotypic change. In posterior cases, 29/40 (72.5%) cases presented in the right eye compared to 10/40 (25%) in the left eye (Table 3). Although the majority of mouse eye phenotypes observed in this screen were anterior in presentation, we were particularly interested in posterior changes that might resemble human retinal diseases. We did not detect any phenotypes that resembled retinitis pigmentosa, nor diabetic retinopathy but did identify some phenotypes that resembled AMD. In the case of mouse line 154.6, a number of white lesions were present that appeared to mimic the drusen seen in AMD and in mouse line 131.27, regions of hypo- and hyper-pigmentation
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Table 3 Abnormalities detected in G1 offspring by eye Group
Type of abnormality
Right
Left
Anterior
Tumescent cataract/ normal size Segment disruption with cataract Microphthalmic Corneal opacity cataract Fleck Cataracts Lid abnormality Subtotal
19
1
2
17
4
0
60 45
6 7
3 8
64 8 213
54 1 73
27 0 40
5 24 29
0 10 10
0 5 5
242
83
45
Posterior
Pole––discs Pole––retina Subtotal Total
Bilateral
were present. Mouse fundi photographs were taken to demonstrate the changes (Fig. 2). 3.6. Mouse lines for breeding A total of 30 G1 mice with different phenotypes of interest were initially selected for breeding in this study to confirm genetic inheritance. These consisted of 25 retinal and five anterior phenotypes. Two mouse lines however did not achieve mating (one posterior and one anterior) due to the old age of the mice. One posterior mouse line died giving birth. Eight of the posterior lines did not produce offspring and breeding was therefore attempted using an assisted reproductive technique of either ‘in vitro fertilization’ (IVF), ‘egg transplantation’, or ‘ovarian transplantation’. In general, IVF was successful (two out of three mice) but egg transplantation and ovarian transplantation ovarian were unsuccessful. This left a total of 17 mice with retinal phenotypes and four mice with anterior phenotypes from which to breed. 3.7. Confirmation of genetic inheritance It was necessary to confirm that the phenotypic abnormality detected was due to an inherited genetic change. As the breeding strategy targeted dominantly induced ENU mutations, a maximum of 50% of offspring should have inherited the causative mutation. Of the 17 retinal phenotypes produced (Table 4), an average of 13 second generation (G2) offspring were produced for each line. With regard to the anterior defects, an average of 8 G2 offspring per line were produced (Table 4). Genetic inheritance was confirmed in both posterior and anterior lines. Two of the four anterior lines produced offspring with the same phenotype as the parent (Table 4). Inheritance in the anterior mouse line 46.15 was 43% (3/7 G2 mice) and in mouse line 19.2, this was
50% (2/4 G2 mice) (Table 4). In the case of the 17 posterior lines, only 3 of the lines appeared to confer genetic inheritance (Table 4). In the case of mouse line 39.7, only 1/12 G2 mice (8%) had an inherited phenotype (Table 4). In mouse line 129.10, this was 2/13 G2 mice (15%) (Table 4) and in mouse line 131.27, 5/14 G2 mice (36%) had an inherited phenotype (Table 4). Of the mice that presented with a similar phenotype to the parent, there did not appear to be any bias in terms of the location within a particular eye (Table 4). In terms of the sex of the G2 offspring, both male and females were represented in the anterior abnormalities (Table 4). In posterior cases, lines 39.7 and 129.10, G2 offspring were only found in female mice although the numbers were small at 1 and 2 mice respectively (Table 4). In the case of mouse line 131.27, all five G2 offspring were in male mice (Table 4).
4. Discussion It is known that a number of retinal phenotypes result from mutational events in single genes, we were interested however in exploring the use of ENU mutagenesis to dissect out components of complex diseases such as diabetic retinopathy and AMD. Our strategy was therefore based on dissecting out single gene mutational events, which on the C57BL/6 genetic background have pleiotrophic effects relevant to retinal disease phenotypes. These mutational events can give rise to either hypomorphic changes (partial loss of function), gain of function or total loss of function (Justice & Bode, 1988). Although some of these mutational events may be rare, complex diseases result as a consequence of many different genes and therefore the probability of detecting one of these genes is increased. In order to try to identify later occurring retinal abnormalities in mice, we chose to perform retinal exams at an age of between 15–20 weeks. It was anticipated that examining mice retina at this age would allow us to detect a broad range of phenotypes whilst still allowing breeding for further genetic studies. Our confidence in this approach was boosted by the identification of a number of mice that presented with retinal phenotypes of interest. All mice with retinal abnormalities were bred to confirm genetic inheritance. The three G1 mouse lines that showed genetic inheritance resulting in posterior changes were followed for disease progression over a period of 18 months. The original eye exam of the G1 mouse line 131.27, defined regions of hypo- and hyperpigmented retinal areas of the right eye and this finding did not change over the next 18 months (Fig. 2). In addition, the other retinal lines examined over this 18month time period did also not appear to progress from the original description given in the eye examination at 15–20 weeks.
P.N. Baird et al. / Vision Research 42 (2002) 479–485
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Fig. 2. Fundus photographs of posterior segment abnormalities: (a) Mouse line 131.27 showing a large white and several smaller whitish areas of retinal hypo-pigmentation and (b) Mouse line 154.6 showing multiple white retinal lesions.
In this study we detected a large number of ocular abnormalities. These included 326 with anterior segment changes and 44 with posterior segment changes of the eye. The predominance of anterior changes agrees with previously published reports on C57BL/6 inbred mice. These changes consisted mainly of microphthalmia, anophthalmia and cataracts (Cook & Sulik, 1986; Robinson et al., 1993); and a low frequency of corneal opacity (Harch, Chase, & Gonsalves, 1978; Cook & Sulik, 1986). Previous reports however have indicated that spontaneous congenital abnormalities occur at between 5% and 15% (Cook & Sulik, 1986; Robinson et al., 1993); whilst in our study these occurred at 28% (326/ 1163). This approximate doubling of the previously reported rate suggests that ENU mutagenesis had led to
this increase in abnormalities. Although we noted a high rate of background phenocopies, our genetic analysis did show inheritance in two lines with cataract/corneal opacity. This again suggested that the introduction of a germline mutation in G0 founder mice had been inherited through G1 and G2 mice (Table 4). The appearance of posterior segment abnormalities, previously not reported, but found in this study, adds to our belief that ENU resulted in mutagenesis. Overall in our study, both anterior and posterior segment abnormalities were 1.23 times more frequent in females compared to male mice. In the case of anterior segment changes, these presented as 180 in females compared to 146 in males and for posterior segment changes, these were 204 in females compared to 166 in
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Table 4 Inheritance of eye abnormality Mouse ID
Sex
Eye
Abnormality
Offspring inheriting abnormality
% inherited
Sex of inherited mouse
Eye of inherited mouse
Anterior 46.15
Female
Right
3/7
43
Female/Male
Left/Right
66.8
Female
Right
0/9
0
31.28 19.2
Female Female
Bilat Bilat
Corneal peripheral haze/post polar cat Microphthalmia/lower lid granuloma Hazy cornea Corneal opacity/ant polar cataract
0/12 2/4
0 50
Female/Male
Left/Right
Posterior 32.11 39.7 147.1 61.8 129.10 13.11 127.8 28.9
Male Male Female Male Male Female Female Female
Left Right Right Right Left Right Left Left
0/13 1/12 0/12 0/14 2/13 0/7 0/9 0/9
0 8 0 0 15 0 0 0
Female
Right
Female
Left/Right
132.4 143.10 168.15
Male Female Female
Right Right Right
145.26 131.27
Female Male
Right Right
Male
Left/Right
19.61 32.56
Female Male
Right Right
154.6 66.13
Female Male
Right Left
Superonasal hypo-pigmentation Disc Cupped disc Nasal hyper-pigmentation Multiple white lesions Raised retina White plaque/subretinal lesion Hypo-pigmented supranasal region Subretinal area of whiteness Pale large disc Hypo- & hyper-pigmented retinal areas Subretinal area of whiteness Hypo- & hyper-pigmented retinal areas Retinal detachment Mottled pigmentary retinal changes White peripheral retinal dots Hypo-pigmented dots
males (Table 2). The only exceptions to this were in the cases of tumescent cataracts and also in lid abnormalities where there was a reversal in the sex bias (Table 2). This alteration of the sex bias compared to previous studies may reflect either a difference in environmental factors or more likely it reflects the use of ENU. We know from our data that ENU does not affect the sex of offspring, as approximately equal numbers of both male and female mice were produced in the study (Table 1). It would therefore imply that either more male mice inherit ENU induced mutations, giving rise to abnormalities, or that environmental factors present in this study resulted in less female abnormalities. This situation could be better clarified if a repeat study was performed on nonENU treated C57BL/6 mice under the same conditions as above. The number of bilateral G1 mice produced as a result of ENU mutagenesis was small in anterior cases and minimal in posterior cases. Anterior bilateral abnormalities were approximately 7.15 times less frequent than unilateral changes (40/213þ73) (Table 3). In posterior cases however, only one of the five G1 mouse lines identified with bilateral retinal changes had a phenotype
In progress 0/5 0/15
– 0 0
0/13 5/14
0 36
0/14 0/13
0 0
0/11 0/15
0 0
of interest but unfortunately died giving birth. The reason for this unilateral bias is unknown but our breeding studies have indicated that mice with unilateral changes do show genetic inheritance (Table 4). Previous reports have also indicated that right eye anterior segment abnormalities are up to 5.8 times more common than left eye abnormalities in C57BL/6 mice (Pierro & Spiggle, 1967). In this study we also found a bias between eyes although only at half the rate previously reported (2.9 times) (Table 3). Genetic inheritance in this study appeared variable with 2/4 anterior segment mouse lines and 3/17 posterior segment mouse lines exhibiting genetic inheritance (Table 4). It was noted that the level of penetrance observed in the mouse lines was variable. In the two anterior lines, penetrance was close to 50% (Table 4) as may be expected for a dominant mutation. In the case of the three posterior mouse lines however the penetrance varied from a maximum of 36% in mouse line 131.27 down to 8% in mouse line 39.7 (Table 4). Most of the 17 G1 mouse lines that presented with retinal changes resembled the early stages of AMD. In the case of mouse line 154.6 multiple white lesions were present that
P.N. Baird et al. / Vision Research 42 (2002) 479–485
appeared to mimic the drusen deposits found in early AMD (Fig. 2). In the case of retinal line 131.27, regions of hypo- and hyper-pigmented retinal areas were seen (Fig. 2), again characteristic of early stages of AMD. The variability in penetrance observed in the 17 G2 retinal lines may reflect the presence of a hypomorphic change where there is only a partial loss of function (Justice & Bode, 1988). It may also reflect the development of a phenotype that occurs later in the mouse lifespan and therefore these mice need to be followed and examined over a longer time frame. This is currently taking place. Further breeding is also underway to produce more G2 offspring from these mouse lines of interest to allow both a better definition of penetrance and also to allow for histological examination. If changes similar to AMD are confirmed following histological examination then further breeding will be conducted to allow genetic linkage studies to be performed and allow identification of the causative gene. The use of ENU clearly provides the ability to generate mouse models of human disease. We have demonstrated that the use of this mutagen is capable of producing mouse retinal phenotypes that resemble human disease. The experimental procedures however can be complicated by the number of spontaneous phenotypic changes that can occur (in the case of anterior segment changes) and the variable penetrance. A welldefined breeding strategy to confirm genetic inheritance is therefore necessary to confirm the penetrance and nature of a causative mutation. We believe that the use of this technique may be of merit in generating new models of mouse retinal disease and hope to extend our findings through the elucidation of equivalent genes involved in human retinal disease.
Acknowledgements We thank all the animal house technicians who took part in the maintenance of animals both at Kew and Parkville campuses of WEHI. This work was supported by a grant from the Ophthalmic Research Institute of Australia and an NHMRC block grant to the Walter and Eliza Hall Institute of Medical Research.
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