Rescue of developmental lens abnormalities in chimaeras of noncataractous and congenital cataractous mice

Hereditary cataracts are usually transmitted as an autosomal dominant trait. Congenital cataracts are a major cause of blindness in children, the prevalence varying from 10-14% (Nelson, 1984). Several cataractous mouse mutants have been used to make detailed studies at the genetic, cellular and molecular levels. In a recent review the advantages of using mouse mutants as model systems to study developing cataracts in humans is discussed (Muggleton-Harris, 1986) . One such mutant is the Cataract Fraser mouse (Car^Fr); the inbred strain CAT is homozygous for the Cat^Fr gene causing congenital cataracts (Muggleton-Harris, Festing & Hall, 1987). The nuclei of the deep cortex fibres of the cataractous lens are pyknotic and vacuolation and degeneration of the cortical fibres occurs. The anterior epithelial lens cells show unusual mitotic activity with the formation of multiple cell layers that infiltrate into the fibres of the lens (Fraser & Schabtech, 1962; Verrusio & Fraser, 1966; Zwaan & Williams, 1968, 1969).

Somatic cell hybridization studies of cultured lens epithelial cells from the cataractous mutant and noncataractous mouse lens epithelial cells (MLE) have shown that the phenotypic in vitro population doubling levels of the cataractous MLE is modified (Lipman & Muggleton-Harris, 1982). These in vitro studies suggested that a modification of the mitotic activity of the Cat^Fr lens epithelial cells and the developmental defects in vivo might be possible. Support for this concept is derived from the work where a rescue of an abnormal developmental defect in the retina (LaVail & Mullen, 1976) and the formation of normal mosaics using (a) ‘lethal’ (t¹²/ t¹²) embryos, and (b) the X-linked lethal jimpy (jpmsd) mutant, had been successfully achieved by the aggregation of normal and mutant embryos to form chimaeras (Mintz, 1964a; Eicher & Hoppe, 1973). A similar approach was used for trisomies 15, 16, 17 and 19, and the results demonstrated that the trisomic cells could contribute to the normal tissues of the developing chimaeric animals (Epstein, Smith, Zamora, Sawicki, Magnuson & Cox, 1982; Cox, Smith, Epstein & Epstein, 1984).

This paper describes how the dominant autosomally inherited congenital lens abnormalities of the CAT mutant are obviated in the majority of live young by forming chimaeric mice from the aggregation of early embryos of CAT and noncataractous DBA/2 mice.

The CAT inbred strain of mouse is homozygous for the Cat^Fr gene causing congenital cataract. This strain had been bred previously at WPI, Worcester, Mass., USA and is now held at MRC Laboratories, Carshalton, Surrey, UK. They are albino and have the strain-specific variant of the glucose phosphate isomerase enzyme, GPI-1B, whereas the DBA/2 noncataractous mouse is GPI-1A (Eppig, Kozack, Eicher & Stevens, 1977). The lens abnormalities of the CAT mutant begin to develop in utero at 15 days of gestation. The inbred strain DBA/2 (noncataractous) mouse carries a dilute brown coat colour, pigmented retina and has no known lens anomalies (Altman & Katz, 1979).

Embryos were obtained from female CAT and DBA/2 mice after natural mating, or superovulation with intraperitoneal injections of 5i.u. pregnant mares’ serum (PMS; Intervet) followed 48 h later by 5i.u. of human chorionic gonadotrophin (hCG; Intervet). The females were paired overnight with CAT and DBA/2 males, respectively, and a vaginal plug taken as an indication of successful mating on the following day. 4- to 8-cell embryos were flushed from the anterior portion of the uterine horns 48 h later with prewarmed (37 °C) Medium 2, containing 4 mg ml^-1 bovine serum albumin (M2 + BSA; Fulton & Whittingham, 1978). The zona pellucida was removed by brief exposure of the embryos to prewarmed acid Tyrode’s solution (Nicholson, Yanagimachi & Yanagimachi, 1975). The zona-free embryos were washed through three changes of fresh medium before further manipulation.

Techniques for producing chimaeras have been described previously (Tarkowski, 1961; Mintz, 1962). In brief, two embryos, one embryo of each genotype, were placed into a small drop of 37°C Medium 16 + BSA (M16 + BSA, Whittingham, 1971) in a 60x15 mm plastic culture dish under paraffin oil, previously equilibrated with medium. Contact between the two embryos in each drop was accomplished with the aid of a glass pipette. Embryos were examined 2–4 h later to establish contiguity. In some instances phytohaemagglutinin (100μgml^-1 in M2, Type E, Sigma) was used for 1-2 min, to assist aggregation of the embryos. The following day the successfully aggregated morulae were transferred into the uterus of pseudopregnant recipient Fj (C57BL/6J female x CBA/Ca male) hybrid females. These females were housed individually and allowed to deliver their offspring.

Hand-held slit lamp (Zeiss) observations were made on the chimaeric CAT↔DBA/2 offspring from 15 to 65 days postnatally. Detection of vacuolation and minor disturbances of the lens fibres, plus abnormal proliferation of the epithelial cells can be clearly detected. However, gross lens abnormalities can be readily detected without the aid of a slit lamp soon after the eyes are open, the lens is smaller (shrivelled) and opaque in comparison with the DBA/2 mouse lens. Comparative observations with control CAT and DBA/2 littermates, as well as CAT↔CAT and DBA/2 ↔ DBA/2 offspring allowed an assessment of lens abnormalities.

Following enucleation of the eye, the retina and lens of the mice were examined by transmitted and reflected light on the stage of a dissecting binocular microscope. A photographic record was kept of the distribution of pigment and clarity of the lens. The lens could then be used for the histological, cellular and biochemical analyses.

After cervical dislocation the eyes were enucleated from control and experimental adult mice and fixed in Camoy’s fluid (3:1 ethanol/acetic acid). The head region of embryos at day 15 of gestation was similarly fixed. After dehydration and paraffin embedding, serial sections (5 μm) of the tissue were cut and stained with haematoxylin and eosin for microscopical examination.

The existence of chimaerism in tissues or cells was assessed by analysis of strain-specific allelic variants of glucose phosphate isomerase (GPI: EC5.3.1.9). These were analysed electrophoretically on small samples of blood, tissue and cells of the donor, controls and experimental chimaeras. The isozymes were separated using a Titan III Zipzone cellulose acetate plate (Helena Laboratories, Beaumont, Texas) with 0·025 M-Tris base, 0·192M-glycine at pH 8·5 for lh at 200 V. Following the application of 2 ml 1·5% agar overlay containing 2-0 ml of lM-Tris-HCl at pH 8·0, 1·0 ml of Imgml^-1 NADP, 0·1 ml of 100 mg ml^-1 fructose-6-phosphate, 0·1 ml of 5·41 g 100 ml^-1 magnesium acetate, 0·lml of 10mgml^-1 dimethyl thiazolyl diphenytetrazolium bromide, 0·lml of 2·5 mg ml⁻¹ phenazine methosulphate and 3 μl of glucose phosphate dehydrogenase, the GPI isozymes could be visualized.

Photographs of the lens and retina pigmentation were taken on the binocular microscope with Pan F film. Histological specimens were photographed with Pan X with a Zeiss photomicroscope.

From a total of 334 aggregations formed between cataract mutant and control embryos (CAT↔DBA/2), 126 coat colour chimaeras were obtained (Table 1). 71 of these had a coat colour ratio distribution of each genotype of approximately 40:60, 50:50 or 60:40. The remainder of the chimaeras obtained showed a wide range in the distribution of each genotype which varied from 70:30 or 90:10 in favour of one genotype or the other as shown in Table 2. At least 61 % of the 71 overt chimaeras who reached 60 days of age had two normal-sized clear noncataractous lenses, and 29% had clear noncataractous lenses, with one, or sometimes both, lenses developing abnormalities after 60 days of age. A small percentage (3%) of the mice had one clear normal-sized noncataractous lens at 60 days of age, and one small congenital cataractous lens. 7 % of the chimaeric mice had congenital cataracts in both lenses (Fig. 1). To control for the methods used for the aggregation technique and embryo transfers, a series of aggregates was made of DBA/2↔DBA/2 and CAT↔CAT embryos. Control, nonaggregated DBA/2 and CAT embryos were also transferred into pseudopregnant recipients. The DBA/2 mice always had clear normal lenses and the CAT mice developed congenital cataracts (Table 2).

The range of coat colour distribution of the chimaeric mice can be appreciated when comparing them with the control DBA/2 or CAT mice in Fig. 2A. The eyes of a chimaera with clear normal-sized lenses can also be compared with those of a congenital cataractous littermate (Fig. 2B).

The histological studies showed a number of differences between the embryonic congenital cataractous lens and the normal DBA/2 lens as can be seen in Fig. 3A,B. The nuclei of the mitotic bow are disturbed. Early vacuolation and swelling of the fibres can be clearly discerned at 15 days gestation. Adult lenses are difficult to section without some tearing of the tissue; however, these artifacts are easy to identify in comparison with the vacuolated pathology of the cataractous lens. Fig. 3C shows the vacuolated and swollen cells of the cortex fibres. Fig. 3D shows the pyknotic nuclei and multilayering cells of the anterior epithelial cells. The cortex fibres are non-aligned, the nuclei of the lens have dissolved and in many places have been replaced by large aqueous filled areas.

Fig. 4A shows the GPI isozyme distribution in a representative sample of 39 clear lenses from adult chimaeric mice that had been rescued from the congenital cataractous state. The lenses were of normal size and slit lamp observations did not detect any defects prior to 60–70 days of age. The ratio of GPI isozymes of both the CAT and DBA/2 geno-types is well represented in a number of the lenses. It is interesting to note that those adult chimaeric lenses that developed lens abnormalities beyond 60 days of age tended to cluster in one area of the GPI distribution pattern, where both CAT and DBA/2 GPI isozymes are well represented (Fig. 4B). In those chimaeras with congenital cataracts no GPI-1A (DBA/2) was detected.

The degree of DBA/2 pigmentation of the retina in the overt chimaeric mice with clear lenses was noted when the eye was enucleated prior to GPI analysis or histological procedures (Fig. 5). There appeared to be an approximate correlation between the ratio of GPI isozymes in the lens and the distribution of pigmented and nonpigmented retina epithelial cells. The degree of pigmentation of the iris and retina could also be seen in the histological sections of the chimaeric lenses. Those lenses that had remained free of any congenital defects had a normal morphology and patches of pigment can be seen in the iris (Fig. 6A,B). Those chimaeras rescued from congenital cataracts but developing lens abnormalities in later life (60-70 days of age) show a number of the typical cataractous lens defects, e.g. disturbed lens fibres, pyknotic nuclei, vacuoles and/or multilayering of the lens epithelial cells (Fig. 6C). A section of the CAT lens at 70 days of age allows a comparison to be made of the abnormal congenital cataractous cell morphology; the pigmentation and normal lens morphology of the DBA/2 mouse can be seen in Fig. 6D,E.

We have produced phenotypically normal chimaeras with significant proportions of the congenital cataractous genotype in a variety of tissues including the lens, retina, also in blood and coat colour. Further-more, the majority of CAT↔DBA/2 chimaeras did not develop lens anomalies that could be detected by slit lamp observations or histological studies. Ultra-structural or biochemical anomalies, if any, did not result in a loss of transparency, biological function or cell regulation. The normal diffraction properties of the lens indicate that the delicate osmotic balance and crystallin synthesis is being maintained by the chimaeric lens cells. Although the lens differentiates from the surface ectoderm by a series of inductive processes culminating in an association with the optic vesicle, the developing lens grows through division of epithelial cells initially throughout the epithelium. Later it is only the germinative zone located in the equatorial region of the epithelium which continues to replicate and the initial cells of the developing lens are retained within the inner cortex fibres of the adult lens. We therefore conclude that the correction of the developing lens congenital defects in the chimaeras occurred during organogenesis. Cells of the lens epithelia, fibres and retina of the embryo and adult chimaera eye contain both CAT and DBA/2 genetic cell markers. This is not surprising in that the melanocytes of the pigmented epithelium are formed from the outer layer of the optic cup which is derived from the optic vesicle.

Control of cell division in the lens remains an important developmental problem and has been shown to be a significant factor in hereditary cataract. The manner in which the regulation of cell replication has been achieved in our experimental chimaeras has not been determined. The data presented in this paper suggest a positive regulating process. Possibilities that may be considered are (1) that there are sufficient numbers of noncataractous cells and/or components in the developing chimaeric lens which induce the congenital cataractous cells to replicate in the normal manner, or (2) that the CAT cells could have been at a proliferative disadvantage and the DBA/2 cells had colonized the tissue. However, our results have shown that the CAT cells form a significant proportion of the adult chimaeric lens. The high proportion of the GPI-1B (CAT) genotype in the clear chimaeric epithelium suggests that the abnormal phenotypic replication associated with the cataractous lens MLE has been modified.

As the noncataractous and CAT embryonic lens cells do not hybridize in vivo, the exchange of cellular components and information would have to be achieved by intercellular communication. The receptors on the cells’ surface or the extracellular matrix may play a role in regulating the cells’ behaviour. We have recently shown that subpopulations of cultured MLE cells can respond to the stimulus of a piece of the collagen lens capsule and synthesize the collagen, glycoproteins and proteoglycan components required for further lens cell differentiation to take place in vitro (Muggleton-Harris & Higbee, 1987).

It has been suggested that gap-junctional communication plays an important role in regulating and coordinating cellular growth (Pitts & Finbow, 1977; Lowenstein, 1979; Sheridan, 1977; Wolpert, 1978; Spray, Harris & Bennett, 1982). Preferential coupling may have taken place in the cells of the chimaeric lens during embryogenesis, and play an important role in regulating cell growth, lens morphogenesis and response to growth factors (Rothstein, Worgul & Weinsieder, 1982; Goodall, 1985). Further data suggesting that a positive regulating process has contributed to the rescue of the experimental lens from the congenital cataractous state are the results that show that the lens epithelial cells of the adult chimaeric lens have a large proportion of CAT cells present. These cells would have proliferated during embryogenesis similarly to the cells of the lens in the dominant congenital cataractous mouse, had their replication not been regulated by the presence of the noncataractous cells and/or their components.

With regard to those adult chimaeras whose lenses developed abnormalities at various times after 60 days of age, the results have indicated that these occur when approximately equal distributions of CAT/DBA/2 components are present (Fig. 4B). Epstein (1985) has recently speculated on a variety of ways in which imbalance of a locus could occur; he suggests for example that changes in the concentrations of gene products, such as rate-limiting enzymes, could play a role. Regulatory molecules, involved in surface recognition and adhesion phenomena, and receptors concerned with inter-cellular communication may disturb the functions of the locus. If such molecules could alter or be diluted with age in the adult chimaeric lens, then lens abnormalities could occur.

The results from these experiments have shown that a rescue from the congenital cataractous state has been achieved in the majority of chimaeras. This rescue must have taken place at the embryonic cellular level, because those cellular defects associated with the congenital cataractous lens are easily detected at the slit lamp and histological level. The unusual mitotic activity of the epithelial cells and vacuolation of the fibre cells, associated with the embryonic CAT lens epithelial cells, would be detected very easily. Also, a high percentage of the chimaeras have a substantial amount of the CAT GPI genotype in their lens tissue, therefore CAT cells form a significant part of the epithelial and fibre cells and yet cannot be differentiated morphologically from the DBA/2 noncataractous lens cells. ‘The ‘rescued’ chimaeric lens differentiates and develops in a normal manner without the dominant congenital cataractous defects occurring.

We wish to acknowledge the partial support for this work by a grant made to A. L. Muggleton-Harris from the National Eye Institute, NIH, USA. We also thank Andy Brammall and Eleanor Rawlings for their technical help.