Lens Rudiment Research Articles

The function of FGF signaling in the lens placode

Previous studies suggested that FGF signaling is important for lens formation. However, the times at which FGFs act to promote lens formation, the FGFs that are involved, the cells that secrete them and the mechanisms by which FGF signaling may promote lens formation are not known. We found that transcripts encoding several FGF ligands and the four classical FGF receptors are detectable in the lens-forming ectoderm at the time of lens induction. Conditional deletion of Fgfr1 and Fgfr2 from this tissue resulted in the formation of small lens rudiments that soon degenerated. Lens placodes lacking Fgfr1 and 2 were thinner than in wild-type embryos. Deletion of Fgfr2 increased cell death from the initiation of placode formation and concurrent deletion of Fgfr1 enhanced this phenotype. Fgfr1/2 conditional knockout placode cells expressed lower levels of proteins known to be regulated by FGF receptor signaling, but proteins known to be important for lens formation were present at normal levels in the remaining placode cells, including the transcription factors Pax6, Sox2 and FoxE3 and the lens-preferred protein αA-crystallin. Previous studies identified a genetic interaction between BMP and FGF signaling in lens formation and conditional deletion of Bmpr1a caused increased cell death in the lens placode, resulting in the formation of smaller lenses. In the present study, conditional deletion of both Bmpr1a and Fgfr2 increased cell death beyond that seen in Fgfr2CKO placodes and prevented lens formation. These results suggest that the primary role of autocrine or paracrine FGF signaling is to provide essential survival signals to lens placode cells. Because apoptosis was already increased at the onset of placode formation in Fgfr1/2 conditional knockout placode cells, FGF signaling was functionally absent during the period of lens induction by the optic vesicle. Since the expression of proteins required for lens formation was not altered in the knockout placode cells, we can conclude that FGF signaling from the optic vesicle is not required for lens induction.

Cataract mutations and lens development.

The lens plays an essential role for proper eye development. Mouse mutants affecting lens development are excellent models for corresponding human disorders. Moreover, using mutations in particular genes the process of eye and lens development can be dissected into distinct steps. Therefore, three mouse mutants will be described in detail and discussed affecting three essential stages: formation of the lens vesicle, initiation of secondary lens fiber cell formation, and terminal differentiation of the secondary fiber cells. The mutant aphakia (ak) has been characterized by bilaterally apakic eyes [Varnum and Stevens (1968) J. Hered. 59, 147-150], and the corresponding gene was mapped to chromosome 19 [Varnum and Stevens (1975) Mouse News Letters 53, 35]. Recent investigations in our laboratory refined the linkage 0.6 +/- 0.3 N cm proximal to the microsatellite marker D19Mit10. The linked gene Pax2, responsible for proper development of the posterior part of the eye and the optic nerve, was excluded as candidate gene by sequence analysis. Histological analysis of the homozygous ak mutants revealed a persisting lens stalk and subsequently the formation of lens rudiments. The lens defects led to irregular iris development and retinal folding. Congenital aphakia is known as a rare human anomaly. Besides a corneal dystrophy (CDTB), no corresponding disease is localized at the homologous region of human chromosome 10q23. The Cat3 mutations are characterized by vacuolated lenses caused by alterations in the beginning of secondary lens fiber cell differentiation at embryonic day 12.5. Secondary malformations develop at the cornea and the iris, but the retina remains unaffected. Two mutant alleles of the Cat3 locus have been mapped to mouse chromosome 10 very close to the microsatellite markers D10Mit41 and D10Mit95 (less than 0.3 cM). Since Cat3 is mapped to a position, which is homologous to human chromosome 12q21-24, the disorder cornea plana congenita can be considered as a candidate disease. The series of Cat2 mutations have been mapped close to the locus encoding the gamma-crystallin gene cluster Cryg [Löster et al. (1994) Genomics 23, 240-242]. The Cat2nop mutation is characterized by a deletion of 11 bp and an insertion of 4 bp in the 3rd exon of Crygh leading to a truncated gamma B-crystallin. The defect in the Crygh gene is causative for the stop of lens fiber cell differentiation from embryonic day 15.5 onward. Besides the lens, no further ocular tissue is affected. The Cat2 mouse mutants are interesting models for human cataracts caused by mutations in the gamma-crystallin genes at human chromosome 2q32-35. The ak, Cat3 and Cat2 mutants are discussed in the context of other mutants affecting early eye and lens development. Additionally, human congenital cataracts are discussed, which have been characterized similar to the mouse models. The overview of the three types of mutants demonstrates that genes, which affect the early eye development, e.g. at the lens vesicle stage, have consequences for the development of the whole eye. In contrast, if the mutation influences later steps of lens differentiation, the consequences are restricted to the lens only. These data indicate a decreasing effect of the lens for the regulation of eye development during embryogenesis.

Developmental patterns of crystallin expression during lens fiber differentiation in amphibians.

Data on activation of crystallin synthesis during lens fiber (LF) formation in amphibians are summarized to point out the questions particularly interesting in the context of lens cell lineage-specific expression programming under different developmental conditions. LFs are known to differentiate throughout life along the same pathway that includes at least five compartments. Using the amphibian eye lens as a model, we have studied how crystallins are expressed in the course of: (1) embryonic LF formation, (2) LF differentiation in adults, and (3) LF transdifferentiation from other (non-lens) eye tissues. Our experiments showed that synthesis of crystallins during morphologically similar LF differentiation in embryonic and adult amphibian lens has different spatial-temporal patterns (i.e., is apparently activated according to different programs). Certain results obtained in our studies suggest the absence of any direct relationship between the capacity of adult newt iris cells to transdifferentiate into LFs and crystallin synthesis (<<molecular predisposition>> to such transdifferentiation) in them. Crystallins appear at the advanced stages of iris transdifferentiation into the lens and dynamics of their synthesis in the <<regenerating>> lens resembles that in the embryonic lens, although a new lens rudiment develops from the adult iris epithelium. Data on alternative patterns of the crystallin gene activation are summarized and compared with recent observations on spatial-temporal expression of Pax genes, which play an essential role in lens cell commitment and crystallin synthesis. On this basis, it is suggested that ontogenetic and tissue- or cell-specific changes in Pax gene expression may result in altered programs for activation of crystallin genes in embryonic, adult, and regenerating lens.

Detection of Ascorbic Acid in the Eye of the Early Chicken Embryo by Silver Staining

Ascorbate is found in high concentrations in the aqueous, lens and retina of a variety of animal species. Several functions have been proposed for this, including a vital role in aqueous secretion. We investigated the possible presence of ascorbate in the early embryonic chicken eye as a marker for the differentiation of the ciliary epithelium. Embryonic chicken eyes were processed for histochemical demonstration of ascorbate by silver staining. A strongly positive reaction was found in the interstitial matrix between the lens rudiment and the optic vesicle of the 2-day chicken embryo. By 3 days, with lens and optic cup separating from each other, the silver grains were concentrated in the vitreous cavity, primarily in a band-like configuration between the lens equator and the rim of the optic cup, which is the presumptive ciliary epithelium. A weaker reaction was seen in the lens vesicle and primitive neural retina. During the second and third day of incubation the optic vesicle is just beginning to expand. The appearance of ascorbic acid in the newly formed vitreous cavity at this time may indicate its importance in the biosynthesis of the matrix that is filling the expanding optic vesicle.

The appearance of α, β and γ crystallins in an anophthalmic strain of mice

A certain percentage of congenitally anophthalmic mouse embryos have the ability to generate small lens vesicles that have previously been shown to produce alpha crystallin at 13-day gestation. Further immunohistological analysis of 13- and 15-day-gestation anophthalmia embryos indicates that beta crystallin is present in those 13-day embryos which have lens vesicles with lens-fiber formation. Also, 15-day embryos with lenses demonstrating fiber elongation can produce both beta and gamma crystallins. The conclusion is drawn that the genetic potential to produce at least three characteristic biochemical markers of normal lens differentiation is present in the anophthalmia mutant. The spatial distribution patterns of the crystallins in normal and anophthalmia embryos were similar. However, there appeared to be a transposition in the temporal appearance of beta and gamma crystallins in the anophthalmia mutant. Optic cups and associated lenses in 15-day anophthalmia specimens were much smaller than those in controls. The optic and lens rudiments in these anophthalmia embryos were fairly proportional in size, which indicates that some degree of allometric growth compensation had occurred during the course of development. This ability for differential growth compensation in the mouse eye appears to be restricted to the predifferentiative stages of eye formation.

Histochemical analysis of extracellular matrix material during embryonic mouse lens morphogenesis in an aphakic strain of mice

Extracellular matrix material (ECM) present during early lens morphogenesis was analyzed histochemically in normal CFW mice and mutant strain aphakia by the Alcian blue 8GX, pH 2.5, Alcian blue 8GX, pH 2.5/periodic acid-Schiff combined, high-iron diamine, and van Gieson methods. At lens placode formation, the optic vesicle basal lamina in both strains was higher in sulfated glycosaminoglycan content than was the ectodermal basal lamina. In the aphakia strain, ECM components were observed intercellularly in the presumptive neural retina and lens rudiment of some specimens. This observation was peculiar to the aphakia strain. At the lens cup stage (10.5 days), the interface ECM became less uniformly dense in the CFW strain, resulting in the formation of a fibrillar structure in the widening interspace area. In contrast, the interface ECM in the mutant strain stained solidly and continuously for acidic materials, particularly sulfated glycosaminoglycans, for a full 2 days longer than in the normal strain. The optic cup and lens rudiment remained closely apposed and intercellular ECM components were observed in these tissues in most mutant specimens throughout these stages. The exact mechanism resulting in these intercellular deposits is unknown, although it is possible that they are either pulled along on the cell surface away from the interface ECM during cell shape changes related to the cell cycle or that they are secreted abnormally due to some disturbed cellular polarity. It is unclear at this time if these abnormalities of the ECM in the aphakia strain play a role in the pathogenesis of the multiple eye anomalies, or if they are a secondary effect of the gene mutation.

The extracellular matrix between the optic vesicle and presumptive lens during lens morphogenesis in an anophthalmic strain of mice

Extracellular matrix material present during early lens morphogenesis in anophthalmic strain ZRDCT-Ch mice was studied histochemically by the Alcian blue 8GX pH 2.5, Alcian blue 8GX pH 2.5/periodic acid-Schiff combined, high iron diamine, and Van Gieson methods. Observed staining patterns were compared with results from an analysis of a normal strain of mice (E. H. Webster, Jr., A. F. Silver, and N. I. Gonsalves, 1983, Develop. Biol. 100, 147–157) . No differences in constituents were found between the strains in staining patterns of the ectodermal basal lamina. However, the optic vesicle basal lamina in the anophthalmic strain was found to have a relatively lower staining intensity for sulfated glycosaminoglycan associated with it than was observed in the normal strain, although these mutant optic vesicles were morphologically normal. Results from this and the earlier study on normal mice indicate that one function of sulfated glycosaminoglycan in early lens morphogenesis may be to serve as a cementing medium between the optic and lens rudiments. This sulfated glycosaminoglycan deficiency on the anophthalmic optic vesicle basal lamina is temporally correlated with and may be causally related to precocious lens cup formation and frequently observed separation of the normally adherent eye rudiments. Conclusions drawn from this study are consistent with the speculation of H. B. Chase and E. B. Chase (1941, J. Morphol. 68, 279–301) that there may be abnormal contact between the optic vesicle and presumptive lens ectoderm in the mutant strain, although there is a differing view on the cause of the abnormal contact.

The mechanisms of cell death and phagocytosis in the early chick lens morphogenesis: A scanning electron microscopy and cytochemical approach

The cell surface characteristics of degenerating cells and phagocytes, as well as the participation of lysosomes in the cell death process associated with the early embryogenesis of chick lens rudiment, were studied by means of scanning electron microscopy and cytochemically using the Gomori-beta-glycerophosphate method for acid phosphatase. The prospective dying columnar epithelial cells lose their apical and basal processes and become rounded. The rounded, isolated, dying cells initially show a rough surface with some cytoplasmic constrictions followed by progressive break-up into several pitted fragments. Coincident with the loss of the columnar cell shape, acid phosphatase is localized within the Golgi apparatus and autophagic vacuoles which progressively increase in size. In contrast, the isolated dying cells and fragments do not show significant acid phosphatase activity. The role of lysosomes in this degenerative process is discussed. Neighboring epithelial cells phagocytose the dead cell fragments, becoming nonspecialized phagocytes. These consist of columnar epithelial cells and free cells which have migrated from the lens epithelium. Two mechanisms of internalization are observed. The most frequent mechanism takes place in both the columnar epithelial cells and the free cells, and consists of the progressive engulfment of the fragments into craters of the cell surface. The other mechanism is only detected in the free cells and takes place by pseudopod engulfment. We suggest that both phagocytic procedures could be related to the degree of intercellular connection. The presence of phagocytic internalization by crater formation in the epithelial cells could be a mechanism preserving the epithelial stability, which is necessary for a normal morphogenesis. Small microprocesses binding the surface of the phagocyte and the fragment are present prior to the internalization process. In the lens stalk and in the space located between the ectoderm and the lens vesicle, there are some cells displaying migratory characteristics. This fact suggests that an active migration of epithelial cells from the lens stalk could account for the process of detachment of lens vesicle from the ectoderm. The free cells appear to undergo an in situ progressive degeneration.

Crystallin synthesis in the lens rudiment of a strain of mice with congenital anophthalmia

Immunofluorescence with anti-crystallin antisera was done on the eye rudiments of mouse embryos of a congenitally anophthalmic strain. In a few of the embryos a lens vesicle formed, which, although much smaller than in controls and delayed in development, appeared morphologically normal. α-Crystallin could be identified in the cells of the rudiments. In addition the cells looked cytologically similar to those of the normal lens vesicle; they were elongated and the nuclei displayed interkinetic nuclear migration. Therefore, the surface ectoderm of the anophthalmia mouse can differentiate into a lens vesicle that appears biochemically and cytologically normal. This indicates that the optic vesicle in this mutant is capable of inducing a lens, while the surface ectoderm can respond to the stimulus in a normal fashion.

The appearance of α-crystallin in relation to cell cycle phase in the embryonic mouse lens

Specific protein synthesis in the embryonic mouse lens was studied by immunofluorescence with antisera to adult mouse lens or crystallin fractions. Positive reactions were first detected in a few cells of the lens cup 18–24 hr after contact between optic vesicle and presumptive lens ectoderm had been established. During formation of the lens vesicle a rapidly increasing fraction of cells produced crystallins. At the time of detachment of the vesicle from the surface all cells of its posterior wall showed immunofluorescence. After fiber elongation became distinct cells of the anterior epithelium began to fluoresce and shortly afterwards the entire rudiment produced crystallins. The early reactions were due entirely to the presence of α-crystallin. Reactions were restricted to the lens. Thus, in the mouse as in other species crystallins were detectable by immunofluorescence in vivo only after lens morphogenesis was well underway and only in the lens rudiment itself. Cells first synthesizing crystallins always had an elongated shape and their nuclei were in a basal position. A few hours later mitotic cells displayed fluorescence. Taking into account earlier found relations between cell morphology and cell cycle phase, this indicates that α-crystallin is first demonstrable in the S-or early G-2 phase of the cell cycle, and that the start of its synthesis does not preclude continued cell replication. It is interesting that the cellular location, cell cycle phase, and developmental stage, in which crystallins first appear, are comparable in mouse and chick embryo. Yet, entirely different proteins are involved: α-crystallin in the first, δ-crystallin in the latter. Implications of this for our understanding of lens induction are discussed.

α-, β- and γ- Crystallins in the regenerating lens of Notophthalmus viridescens

Removal of the lens from the eye of adult Notophthalmus viridescens, the Eastern spotted newt, is followed by regeneration of another lens from the dorsal iris. This cell-type conversion of iris epithelial cells into lens cells is accompanied by the subsequent synthesis of α-, β- and γ-crystallins, proteins specific for the normal vertebrate lens. The immunofluorescence technique was employed to determine the ontogeny and localization of the crystallin classes in the regenerating lens rudiments. Antibodies specific for α-, β- and γ-crystallins were applied to tissue sections through regenerate Stages III–XI Yamada 1967. The first positive reaction for α-crystallins did not occur until Stage VII, in a few cells of the developing lens fiber region; and in the external layer (presumptive lens epithelium) at Stage VIII, at which time secondary lens fibers have begun to form. β-Crystallins were first detectable in the thickening internal layer of the Stage V lens (vesicle) and in the external layer at Stage VIII. The γ-crystallins also first appeared, albeit erratically, in a few cells of the internal layer of the Stage V regenerate, and were not detectable in the external layer/lens epithelium at any time. Thus the crystallins are not present in the earliest stages, but are indicative of cellular differentiation in succeeding stages of lens regeneration.

A quantitative study of the position of the Golgi apparatus in the early developing chick eye.

A quantitative evaluation of the positional changes of the Golgi apparatus during the invagination of the lens placode and the presumptive neural retina of the chick embryo was carried out by silver impregnation. The Golgi apparatus is predominantly polarized in the apical process of the elongated interphasic cells; however, basal and lateral positions are also present. When the nuclei are located close to the luminal surface, basal and lateral positions increase significantly showing that a loss of Golgi apparatus polarization takes place, associated with the mitotic cycle. At the beginning of the invagination of lens and neural retina rudiments, a marked shift in orientation occurs between lateral and apical positions of the Golgi apparatus; this is associated with the nuclei located close to the luminal surface. The possible significance of these results is related to some processes concerning the invagination of these rudiments, such as interkinetic nuclear migration and secretion of extracellular materials.

Embryonic Appearance of α, β, and γ Crystallins in the Periodic Albinism (a p) Mutant of Xenopus laevis

The appearance of the crystallins during lens development in the periodic albinism (ap/ap) mutant of Xenopus laevis has been studied. Using antibodies specific for total crystallins, alpha + beta crystallins, and gamma crystallins in the immunofluorescence technique, the first positive reaction for all could be demonstrated in the Nieuwkoop-Faber Stage 31 lens rudiment. The antibody to alpha + beta crystallins exhibited differences in intensity from cell to cell in the early rudiment, while the reaction to the other antibodies was uniform throughout the rudiment. As lens differentiation progressed, immunofluorescence was restricted in all cases to the lens fiber area, up to and including Nieuwkas positive, however, for total lens crystallins. These results are at variance with earlier studies on lens development and the crystallins in wildtype (+/+) X. laevis, where a positive reaction for gamma and total crystallins could be detector total lens crystallins. That this divergence in the mutant is due to a pleiotropic effect or directly to the inductive failure of the endomesoderm to initiate melanogenesis, is discussed.

Cell death during detachment of the lens rudiment from ectoderm in the chick embryo

The distribution and cytological characters of physiological cell death associated with formation of the lens vesicle in the chick embryo was studied by means of vital staining, light and electron microscopy. We have established a constant pattern of cell death which differs in some aspects from that reported for mammals and amphibians. The necrotic process is observed initially in the dorsal part of the lens cup (stage 15). The necrotic area progressively surrounds the lens pore as invagination proceeds (stage 16) and at stage 17 is located in the zone of fusion of the invaginated lens. After detachment of the lens (stages 18, 19 and 20) dead cells appear in the ectoderm, in the superficial epithelium of the lens vesicle and in the space between both structures. Ultrastructurally we observe isolated dead cells in different stages of degeneration and in phagocytosed cells. Phagocytosis is carried out by the neighboring healthy epithelial cells. Phagocytic activity was detected concomitant with the beginning of the necrotic process. Cell fragments were occasionally detected within the lens cavity.

Ontogeny and localization of the crystallins during lens development in normal and Hy-1 (hyperplastic lens epithelium) chick embryos

A strain of chickens selected for high growth rate has been found to exhibit an anomalous eye lens morphology indicating a failure of the normal process of growth regulation. Hyperplasia of the lens epithelium and annular pad, often with fiber formation, has recently been described in day-old chicks of this strain, termed Hy-1 (Clayton, 1975). The earliest evidence of this condition in this study has been found in the 11-day embryonic Hy-1 lens. Before this time, no definitive lens abnormality could be detected histologically in the Hy-1 embryos. However, the indirect immunofluorescence technique had revealed early temporal and spatial differences with regard to the lens crystallins. Antibodies, specific for delta, cathodal beta, anodal beta and alpha crystallins, were applied to sections through the lens of 2 1/2-, 3-, 3 1/2, 4-, 5-, 8- and 16-day embryonic and 1-day post-hatch normal and Hy-1 chicks. delta crystallin appears precociously in the external layer, and alpha crystallin in the prospective fiber region, of the lens rudiment of Hy-1 embryos. Both anodal and cathodal beta crystallins are retarded, however, in their appearance in the external layer/epithelium of Hy-1 lenses. Localization of the crystallin classes within the lenses of the two strains continues to vary during lens differentiation until 1 day post-hatch, at which time and during late embryogenesis annular pad and epithelium abnormalities can be frequently be seen in the Hy-1 lens. This inability to control normal lens histogenesis thus manifests itself early as alterations in the appearance of an organ-specific gene product, the crystallins.

Eye lens development and γ crystallins in Discoglossus pictus (Anura)

The ontogeny and localization of the gamma crystallins in Discoglossus pictus lens development has been determined. Using antibody specific for amphibian gamma crystallins in the immunofluorescence technique, it was found that gamma crystallins first appear in primary lens fibre cells in the lens rudiment, and continue to be restricted to the fibre area as lens development progresses. Thus the role of gamma crystallins as indicators of a differentiated state remains constant in amphibian evolution, having been demonstrated in the most archaic anuran superfamily, as well as in others more recently evolved.

Quantitation of delta-crystallin messenger RNA during lens induction in chick embryos.

Embryonic chick cells of the presumptive lens ectoderm are induced to differentiate into a lens by the optic vesicle, an outgrowth from the developing brain. This lens induction involves formation of the lens placode by cell elongation between about 44 and 50 hr of development, followed by invagination of the placode between about 50 and 55 hr of development. The amount of delta-crystallin messenger RNA (mRNA) in heads of embryos between 48 and 72 hr of development was determined by molecular hybridization with [3H]DNA complementary to purified delta-crystallin mRNA. An average of approximately 0.5 pg of delta-crystallin mRNA was found per embryonic head at 48 hr of development. This leads to approximately 265 molecules of delta-crystallin mRNA per lens cell, if one assumes that the mRNA is confined to the lens rudiment, as is indicated by immunofluorescence studies of delta-crystallin performed by other investigators. Pulse-labeling experiments with [35S]methionine indicated that the delta-crystallin mRNA is being translated already at this time. delta-Crystallin mRNA accumulated at an average rate of 1 molecule per cell per minute between 48 and 72 hr of development. Thus, by extrapolation, the initiation of delta-crystallin mRNA accumulation coincides with the initiation of lens placode formation at approximately 43-44 hr of development, which is some 8-9 hr after the initiation of lens induction by the optic vesicle. These data suggest that delta-crystallin synthesis is regulated by the accumulation of delta-crystallin mRNA during lens induction in chick embryos.

The matrix of the optic vesicle-presumptive lens interface during induction of the lens in the chicken embryo

The cell coats of the presumptive lens cells and the extracellular interface between the lens rudiment and optic vesicle were investigated in the chicken embryo throughout the period during which lens induction is presumed to take place. Histochemical methods showed that the cell coats contained both glycoproteins and glycosaminoglycans. Autoradiography after [3H]glucosamine injection indicated incorporation of the precursor with subsequent localization primarily at the cell surface. No obvious changes in the properties of the coat were noted with the progression of early lens morphogenesis. The extracellular matrix at the interface between ectoderm and optic vesicle also contained glycoprotein and glycosaminoglycan. There was a heavy concentration of [3H]glucosamine-containing macromolecules in the area. Electron microscopy revealed that the interface consisted of the basement membrane systems of lens and optic vesicle, fused with their external fibrillar layers. In contrast to the findings on cell coats the density of the interfacial matrix increases appreciably during the lens induction period. Evidence suggests that the cells of the two ocular epithelia are themselves the source of the matrix materials. It is proposed that the macromolecules excreted by the epithelial cells into the interface interact at different concentrations to form aggregates of various structure by a process of self-assembly. This may be reflected in the different ultrastructure of the layers of the interfacial matrix. Quantitative changes in the density of the matrix, leading to increased adhesion between lens rudiment and optic vesicle, may restrict the lateral spreading of the lens cells and so fix the basal area of the lens rudiment. This, together with continued cell replication, may produce the cell crowding, placode formation and invagination characteristic of lens morphogenesis.

Malorientation of mitotic figures in the early lens rudiment of aphakia mouse embryos.

In early embryonic epithelia most cell divisions are oriented with their cleavage furrows perpendicular to the free surface of the tissue. This results in the generation of two daughter cells, which are equivalent with regard to their apical surface. Mitotic figures, in which the long axis through the centers of the chromosome plates (chromatin masses) is perpendicular to the surface, are seen only occasionally. The lens placode and early lens cup of mouse embryos homozygous for the gene aphakia show significantly greater numbers of maloriented mitoses than normal control lenses. The axis connecting the centers of the chromosomal plates is frequently perpendicular or oblique to the surface. This may result in the production of daughter cells, which are unequal as far as their position at the tissue surface is concerned. Later, the lumen of the mutant lens vesicle fills up with cells, which are apparently released from the lens epithelium. Further development of the lens and the eye is grossly disturbed. The release of these cells may be a consequence of the malorientation of their divisions, or both may be the result of a defect in the spatial organization of the lens tissue, possibly due to abnormal cellular adhesion.

A RADIOIMMUNOASSAY FOR THE DETECTION OF DELTA CRYSTALLIN IN CHICK LENS DIFFERENTIATION.

Delta crystallin was isolated from 10-13 day chick embryo lens fiber cells. The lens fiber cell extract was isoelectrically precipitated at pH 5.1 to remove alpha and beta crystallins. Further purification by filtration through Sephadex G-150 and then acrylamide gel electrophoresis yielded a single, homogeneous preparation of delta crystallin, as characterized by gel electrophoresis. This purified delta crystallin was injected into rabbits to produce a potent antiserum to chick lens delta crystallin. The purified delta crystallin was iodinated with 125 Iodine, using the chloramine-T procedure. A radioimmunoassay for delta crystallin was then developed, using the principles of competitive protein binding analysis. The radioimmunoassay developed here had a minimum sensitivity of 50 nanograms, and effectively ranged to 1000 nanograms. Developing lens rudiments from early chick embryos, beginning from 24 hr incubation up to 72 hr were examined at 6 hr intervals. All determinations from 24 hr through the 48 hr sample showed less than 10 nanograms per 100 lens rudiments. This was below the effective minimum detection limits of the assay. The first accumulation of delta crystallin was detected in the 54 hr sample, and increased thereafter.