Lens Differentiation in Vertebrates: A Review of Cellular and Molecular Features

Chapter 3 – The Lens: Development, Proteins, Metabolism and Cataract

The eyes of amphibians, both anurans and urodelans, have interested students of development since the beginning of the twentieth century. Spemann investigated the roles of the optic vesicle and surface ectoderm in lens formation (Spemann, 1903, 1905, 1907, 1912, 1938) before he began his better known studies on neural induction and the “organizer.” In the United States, W. Lewis (1904, 1907a, b) and Harrison (1920, 1929) also employed this system of lens induction in their work on dependent and independent development. Mangold (1931) wrote a very complete review of the experiments on amphibian eyes up to that time. These early investigations consisted of the following types of microsurgical experiments: 1. isolating the prospective lens-forming ectoderm by removing the eye-forming region of the neural plate or the optic vesicle, 2. combining optic vesicle and ectoderm of different prospective significance located in other regions of the head or flank, and 3. transplanting the prospective lens ectoderm to other parts of the body. Several different species were employed and these were found to differ in the ability of the prospective lens ectoderm to differentiate into a lens without an eyecup and in the capacity of the optic vesicle to induce a lens from foreign ectoderm. Additional investigations seemed to indicate that these properties varied inversely in different species and led Spemann (1938) to propose the hypothesis of “double assurance” in lens formation. This concept postulates two processes, each of which can lead to lens development under experimental conditions: 1. self-differentiation of prospective lens ectoderm, and 2. lens induction by the optic vesicle. In normal development, these two mechanisms reinforce each other. In a later section of this account, it will be seen how recent investigations have led to refinements in our knowledge of the interactions leading to the development of a lens in its proper relation to the eyecup.

The Amphibian Eye: Development and Regeneration

A quantitative analysis of cell division and cell elongation was carried out during lens morphogenesis in the rat. At 13 days of development elongating cells in the posterior part of the lens vesicle (presumptive fibre cells) have a lower mitotic activity than cells in the anterior vesicle. By 14 days these elongating cells do not divide. Thus at 14 days of development the lens can be separated into two compartments; a proliferation compartment in the anterior lens and an elongation compartment in the posterior lens. The three main groups of lens-specific proteins, α-,β- and γ-crystallins, were localized by immunofiuorescence. Alpha-crystallin is the first crystallin to be detected and is localized in some lens pit cells at 12 days of development. By 14 days all lens cells contain α-crystallin. Beta- and β-crystallins are detected later at 12½ days and are localized in some cells situated primarily in the posterior part of the lens vesicle. At later stages of development these crystallins are restricted to cells of the elongation compartment, i.e. presumptive fibre and fibre cells. Possible mechanisms that govern the temporal and spatial distribution of crystallins are discussed.

Cell division, cell elongation and the co-ordination of crystallin gene expression during lens morphogenesis in the rat

Previous studies have shown that cells in the 6-day old embryonic chick lens epithelium elongate in tissue culture. In the present study, the time course of elongation during the 1st day of cultivation has been examined histologically. Cultured epithelia were also treated with cycloheximide or colchicine in order to determine if cell elongation depends on new protein synthesis and on the utilization of microtubules, respectively. In the first 5 hr of culture, the mean cell length increased from 11 µ to 21 µ. Subsequently, elongation was slower; the mean cell length was 28 µ after 24 hr in culture. Continuous exposure to cycloheximide did not inhibit the initial doubling of cell length, but did prevent further elongation. By contrast, colchicine inhibited elongation almost immediately. When added after the cell length had doubled, cycloheximide and colchicine each inhibited further elongation; the treated cells remained columnar. Radioautographic and electrophoretic tests showed that protein synthesis was not appreciably affected by colchicine, but was suppressed by cycloheximide. Electron microscopic examination revealed that microtubules oriented along surface membranes were present in epithelia cultured with serum alone and with cycloheximide, but not in those incubated with colchicine. These results indicate that the early stages of cell elongation in the cultured lens epithelium require an initial assembly and organization of preexisting microtubular elements and that continued elongation depends, in addition, on the de novo synthesis of protein, possibly microtubule protein.

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Isaura Meza

Papers

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