Immunofluorescence localization of the crystallins in amphibian lens development, with special reference to the γ-crystallins

Abstract

The ontogeny and localization of the crystallins in the developing lens of R. pipiens has been investigated by means of the immunofluorescence technique. Antibodies to adult R. pipiens total-lens proteins and adult R. pipiens γ-crystallins—whose antigen, tissue, and developmental specificity had been demonstrated—were used in both the direct and indirect (or “sandwich”) immunofluorescence techniques; both methods gave comparable results. These antibody-dye conjugates were applied to sections of embryonic R. pipiens; a series of lens developmental stages was recognized and defined by us to aid in identification of those stages subjected to immunofluorescence analysis. A positive reaction for the anti-total-lens-protein immunofluorescence reagent was first noted at Lens Developmental Stage VI, in a number of prospective primary lens fiber cells, and a weak, irregular reaction was also noted in the external layer. The intensity of immunofluorescence with this reagent increased in the fiber area and external layer until at Lens Developmental Stage X all cells of the lens were positive; the primary and secondary lens fiber cells and lens epithelium exhibited immunofluorescence in order of decreasing intensity. The first positive reaction with the anti-γ-crystallin immunofluorescence reagent could also be observed at Lens Developmental Stage VI. However, the immunofluorescence was present only in the region of the prospective primary lens fiber cells and was absent in the external layer. The intensity of the immunofluorescence increased in the fiber area until Lens Developmental Stage X, at which time it was greater in the primary fiber cells than that in the secondary fiber cells. No immunofluorescence could be detected in the external layer or in the lens epithelium with this reagent until Stage X. At this time, after the lens is well differentiated and has assumed the basic adult configuration, a weak immunofluorescence reaction was noted in the lens epithelium in a small percentage of cases. At no time, with either immunofluorescence reagent, could an immunofluorescence reaction be observed outside the lens tissue. Such results indicate that the γ-crystallins, previously implicated in bovine and regenerating newt lens fiber differentiation, are also indicative of lens fiber differentiation in the normal, developing lens of the anuran amphibian, R. pipiens. In addition, the relatively late appearance in lens development (i.e., after formation of a lens vesicle) of any immunofluorescence reaction for the crystallins suggest that they are not necessary for lens induction and subsequent lens placode formation.