The Differentiation of the Crystalline Lens

0 From the Department of Anatomy and Embryology, Faculty of Medicine, University of Amsterdam T H E first sign of lens development in the vertebrate embryo is the appearance of a thickening of the head ectoderm in the area of contact between the ectoderm and the eye vesicle (lens placode). The cytoplasm of the placode cells in the chicken embryo loses its vacuolization, the cells acquire a cylindrical form, the nuclei arrange themselves perpendicularly to the contact surface and move to the base of the cells (McKeehan, 1951). At about the same time chemical changes take place in the placode cells. Ten Cate and Van Doorenmaalen (1950), using serological methods, were able to demonstrate the presence of specific lens proteins in the placodal stage of the lens in axolotl and chicken embryos. They could not entirely exclude the possibility of the presence of these proteins in the ectoderm at earlier stages, but with their sensitive method they got no indication of them before the placodal stage. At present it is, therefore, impossible to decide whether the appearance of this early protein specificity must be regarded as a pre-morphological cell differentiation or whether it occurs synchronously with or after the morphological changes described by McKeehan. One is inclined, however, to assume that processes of chemodifferentiation by which the typical building-blocks of the cells are produced will precede visible morphological changes. It is generally accepted that the changes in the head ectoderm are due to an influence which the eye vesicle exercises upon that ectoderm at the contact surface (embryonic induction). One might consequently assume that the production of specific lens proteins takes place under the influence of an inductive action of the eye vesicle. To investigate this assumption the following experiments were done. Saline extracts of presumptive lens ectoderm of axolotl neurulae (before the appearance of a lens placode) were mixed with rabbit anti-serum against lens proteins. No precipitin reaction could be demonstrated. Neither could a precipitin reaction be found when saline extracts of young eye vesicles (freed from head - ectoderm) were mixed with the anti-serum. When, however, ectoderm extract and eye vesicle extract were mixed and incubated at 37 C. for 24 hours the mixture, when tested with the anti-serum, showed a precipitin reaction whereas the separate components of the mixture did not contain antigens after the same incubation (Woerdeman, 1950). Thus it seems permissible to assume that the production of specific lens proteins must be ascribed to an interaction between head ectoderm and the eye vesicle. It is, of course, probable that this protein production takes place in the ectodermal cells normally as a very slow reaction and is only accelerated by substances passing from the eye vesicle into the ectoderm, but it is also imaginable that it would not occur at all without the influence of the eye vesicle and that enzymes of the cytoplasm of the ectodermal cells have to be activated or coenzymes to be delivered by the eye vesicle. Substances may also pass into the ectoderm which together with other ectodermal substances may be combined to form lens proteins. Still another possibility is that lens proteins may be present in the ectoderm in a masked form and be unmasked. It seems anyhow to be proved that their demonstrability in the lens placode of amphibians is a result of an action of the eye vesicle on the surface ectoderm. The objection can be raised that in some amphibians (e.g. Rana esculenta, Spemann, 1912; Xenopus laevis, Balinsky, 1951) the lens can apparently develop independently when the eye vesicle is absent. In Rana esculenta, however, it is very probable that lens induction occurs at a very early stage (open neural plate) and takes only a very short time (Woerdeman, 1952). The same may be the case in Xenopus. I see therefore no reason to admit that some amphibians must be excepted from the general rule that lens formation is produced by an induction process. When lens placodes of young frog neurulae are extirpated and grafted under the ectoderm of the abdominal wall or when they are explanted into saline solutions, they develop into small undifferentiated vesicles. In some cases, however, some of their cells show a transformation into lens fibres, but this happens only when somewhat older donors are used (Woerdeman, 1941). When undifferentiated lens vesicles are transplanted in the manner mentioned above, they develop into small lenses with an irregular mass of lens fibres. There are indications to show the presence of a very short period during which no difference in fibre-forming potency exists between the exterior and the interior wall of the lens vesicle, but very soon fibre formation originates only from the interior wall which has been in contact with the presumptive nervous layer of the retina. These observations, together with many data from the literature on lens development, indicate that for a normal differentiation of the lens a lasting influence of the eye cup on the lens anlage is necessary. Most of my observations mentioned above were made on larvae of Rana esculenta, and here again the objection may be raised that in Spemann's paper of 1912 well-differentiated lenses are to be seen which have developed independently of an eye cup. In Spemann's laboratory the eggs were often kept at a low temperature before the operation. Ten Cate (1946) showed that under these circumstances the chemodifferentiation is less retarded than the morphological processes. Before the cooled embryos reach the stage of operation, induction may have taken place during a much longer time than under normal conditions, and chemo-differentiation may have proceeded almost normally. This may explain why, after extirpation of the eye anlage in cooled neurula stages, a well-differentiated lens develops, whereas it fails to develop if the same operation is carried out on neurulae kept at room temperature. If this explanation is valid Rana esculenta would develop a lens in accordance with what has been shown for other amphibians and no discordance would exist between the result of my experiments on Rana esculenta (at room temperature) and the results of Spemann. Our conclusion must therefore be that the induction process in amphibians is a process of long duration. It takes only a short time to start the formation of specific lens proteins in the ectoderm, and the development of a placode (the first visible morphogenetic phenomenon). When the inductive action continues, the placode acquires the potency of vesicle formation and of separating itself from the surface ectoderm. In a following phase a change takes place in the interior wall which enables its cells to form fibres (development of a medio-lateral polarity). It is still questionable if all these differentiation steps are caused by one inductive factor. It might be assumed that there is (...truncated)