Reinvestigation of the role of the optic vesicle in embryonic lens induction

ROBERT M. GRAINGER 0 JONATHAN J. HENRY 0 ROBERT A. HENDERSON 0 0 Department of Biology, University of Virginia , Charlottesville, VA 22901 , USA - The induction of the lens by the optic vesicle in amphibians is often cited as support for the view that a single inductive event can lead to determination in a multipotent tissue. This conclusion is based on transplantation experiments whose results indicate that many regions of embryonic ectoderm which would normally form epidermis can form a lens if brought into contact with the optic vesicle. Although additional evidence argues that during normal development other tissues, acting before the optic vesicle, also contribute to lens induction, it is still widely held, on the basis of these transplantation experiments, that the optic vesicle alone can elicit lens formation in ectoderm. While testing this conclusion by transplanting optic vesicles beneath ventral ectoderm in Xenopus laevis embryos, it became apparent that contamination of optic vesicles by presumptive lens ectoderm cells can generate lenses in these experiments, illustrating the need for adequate host and donor marking procedures. Since previous studies rarely used host and donor marking, it was not clear whether they actually demonstrated that the optic vesicle can induce lenses. Using careful host and donor marking procedures with horseradish peroxidase as a lineage tracer, we show that the optic vesicle cannot stimulate lens formation in neurula- or gastrula-stage ectoderm of Xenopus laevis. Since the general conclusion that the optic vesicle is sufficient for lens induction rests on studies in many organisms, we felt it was important to begin to test this conclusion in other amphibians as well. Similar experiments were therefore performed with Rana palustris embryos, since it was in this organism that optic vesicle transplant studies had originally argued that this tissue alone can cause lens induction. Under conditions similar to those used in the original report, but with careful controls to assess the origin of lenses in transplants, we found that the optic vesicle alone cannot elicit lens formation. Our data lead us to propose that the optic vesicle in amphibians is not generally sufficient for lens induction. Instead, we argue that lens induction occurs by a multistep process in which an essential phase in lens determination occurs as a result of inductive interactions preceding contact of ectoderm with the optic vesicle. The first experimental demonstration that the formation of one embryonic tissue depends on its interaction with an adjacent tissue was performed by Spemann (1901), who showed that lens formation is dependent on an interaction with the eye rudiment. Normally the presumptive lens ectoderm first comes in contact with eye tissue when the latter is formed as an outgrowth of the forebrain just after neural tube closure. Lens differentiation commences shortly after this stage. The cells in the presumptive lens region first become elongated, then invaginate and finally pinch off to form a lens vesicle inside the eye cup. Spemann showed that the optic rudiment was necessary for lens development in the amphibian Rana temporaria (known originally as Rana fusca) since ablation of this rudiment blocked lens formation. Lewis (1904, 1907) reported shortly thereafter that in Rana palustris and Rana sylvatica one could move the optic vesicle beneath nonlens ectoderm and stimulate lens formation in this tissue. The experiments of Spemann and Lewis, and numerous similar studies that followed (reviewed by Reyer, 1958a), introduced what has now become a widely cited idea: that the specification of cell fate in a multipotent tissue can be controlled by a single inductive interaction. There is considerable evidence that, besides the optic vesicle, other inductive effects are also important in lens formation. King (1905) and many others subsequently (Reyer, 1958a) have shown that small lenses or lens-like structures (lentoids) can form in some amphibian species even when the optic rudiment is removed from young embryos. Arguments have been presented (Liedke, 1942, 1951; Jacobson, 1958, 1966) that the formation of these 'free' lenses is the result of interactions of head ectoderm with endoderm and mesoderm underlying it during gastrulation and early neurulation. Thus, there is evidence that important inductive effects are caused by these tissues as well as by the eye vesicle. All of these studies taken together suggest that inductive effects from either source can be sufficient to elicit some degree of lens formation and that the relative effects may vary from species to species. In this paper, we reexamine the view that the optic vesicle alone can cause lens induction in ectoderm and consider whether such a single-step model for induction accurately represents a mechanism for determination in this system. Our first experiments involved optic vesicle transplants in Xenopus embryos. It became clear that an unambiguous method for distinguishing host and donor tissues was required to determine whether lenses found in association with eye tissue in these experiments were induced from host ectoderm or were derived from presumptive lens ectoderm cells which are difficult to remove from donor optic vesicles. Our results indicated that optic vesicles from which all presumptive lens ectoderm is removed cannot elicit lens formation in neurula flank or belly ectoderm of Xenopus laevis. This finding stimulated two directions of investigation. It raised concern about the lens-forming potential of ectoderm in Xenopus at various developmental stages and led to a systematic analysis of this problem (Henry & Grainger, 1987). It also led us to question the sufficiency of the optic vesicle for lens induction among amphibians in general since careful host and donor marking has rarely been used in such studies (Spemann, 1908, 1912; Stone & Dinnean, 1943; Liedke, 1951). We therefore undertook a more extensive series of transplantation experiments, presented here, with Xenopus and, in addition, with Rana palustris since this was the organism used in the first such transplantation experiments. Under conditions similar to those used in the original studies using Xenopus and Rana palustris (Brahma, 1959; Lewis, 1904), we find, contrary to these reports, that the optic vesicle cannot elicit lens formation in ventral ectoderm. Based on this data, we propose that the optic vesicle is generally not sufficient for lens induction and that, therefore, early inductive effects are an essential part of a multistep process for lens cell determination in amphibians. Materials and methods Embryos Embryos were obtained from Xenopus laevis as described by Henry & Grainger (1987). Rana palustris embryos were collected from local ponds. Xenopus and Rana embryos were raised in 20% Steinberg's solution (Rugh, 1962) containing 100 units mP1 streptomycin, 100 units ml"1 penicillin and 25figm\~l (...truncated)