Permissive and directive interactions in lens induction

By MARKETTA KARKINEN-JAASKELAINEN 0 1 0 Author's address: Third Department of Pathology, University of Helsinki , Finland 1 From the Third Department of Pathology, University of Helsinki , Finland The interactive events leading to lens formation and the developmental potentialities of the presumptive lens ectoderm were examined in vitro. The presumptive lens ectoderm of both mouse and chick embryos was capable of forming a lens even when isolated from the optic vesicle before the two tissues reach the stage of close association. This lens-forming bias can be released with favourable culture conditions and by various heterotypic mesenchymes. The same permissive, unspecific conditions or heterotypic tissues failed to trigger lens formation in trunk ectoderm. The directive effect of the optic vesicle was demonstrated in experiments where it was grown in contact with the trunk ectoderm. The latter developed distinct lentoid bodies synthesizing lens proteins. The origin of the lentoid was confirmed in interspecies combination of chick and quail tissues. It is concluded that lens formation is governed by a series of interactive events consisting of both directive and permissive influences. - varying from one species to another, and also depending on the temperature at which the embryos are reared. At lower temperatures, chemical differentiation proceeds more rapidly than morphogenesis (Twitty, 1928). Embryos kept in the cool before experiments will form lenses much more readily than those kept at room temperature throughout development (Ten Cate, 1953). Several researchers failed to obtain differentiation from the presumptive lens ectoderm in vitro on species where it is possible even in the absence of the retinal anlage in vivo (Perri, 1934; Woerdeman, 1941; de Vincentiis, 1949; Jacobson, 1958). It was suggested that lens differentiation depends on the inductive influence of some other tissue besides the optic vesicle (Mangold, 1931; Liedke, 1951, 1955). Okada & Mikami (1937) substituted several tissues in the place of the optic cup in Triturus pyrrhogaster and were able to induce a lens with the following: nose anlage, ear vesicle, brain, heart, liver, and from younger embryos dorsal archenteron wall, neural plate, ectoderm, mesoderm, and entoderm of the head region. Lens formation may also be elicited by unspecific triggers, such as salamander liver, boiled salamander heart (Holtfreter, 1934) or alcohol-treated liver, a known inductor of anterior central nervous system structures (Toivonen, 1949), even by treatment with acetone or alcohol, as in Fundulus embryos (Werber, cited in Twitty, 1955). During normal development, the first tissue to underlie the amphibian presumptive lens is the entodermal wall of the future pharynx. While gastrulation proceeds the edge of the mesodermal mantle, the future heart, extends to the posterior margin of the lens ectoderm. During the neurula stage the neural folds lift the lens ectoderm from contact with the mesoderm and the future retina evaginates from the wall of the neural tube as the optic vesicles, which approach the ectoderm, making contact with the presumptive lens cells. All three tissues, the pharyngeal entoderm, the heart mesoderm and the optic vesicle, are potent lens inductors (for review, see Jacobson, 1966). The entoderm and mesoderm gradually lose their inductive capacity, but the retina does not. As the lens grows throughout life, it continuously requires the presence of the inductor. All 'free' lenses, which differentiate for some time even in the absence of a retina, eventually degenerate. Although Jacobson (1956) predicts that the process of lens induction is similar in all vertebrates, the only difference being in timing of the response of the target ectoderm and hence in the degree of dependence upon the neural inductor, our knowledge concerning the higher vertebrates is fragmentary. Optic vesicle-dependent lens formation has been described in experiments both in chick (Waddington & Cohen, 1936; Alexander, 1937; van Deth, 1940; McKeehan, 1951; Langman, 1956) and in mouse (Muthukkaruppan, 1965). Alexander (1937) and van Deth (1940) also reported that body ectoderm from avian embryos 2 days old or younger responded to the inductive stimulus of the optic vesicle by forming a lens. With advancing age of the host embryos, the capability for lens formation was gradually lost as the cells of the posterior body ectoderm lost their responsiveness early, while those of the head ectoderm retained it for some time. McKeehan (1951) reported lens formation in 4-somite embryos, not only in the head region ectoderm but also in the extra-embryonic ectoderm. The inductive influence of the cephalic endomesoderm of early chick embryos was studied by Mizuno (1970, 1972). He suggested that lens induction is a twostep process, in which the hypoblast with some mesoblast cells first acts upon the undermined epiblast until the streak stage, whereafter lens formation can be experimentally triggered by several other tissues than the optic vesicle, such as embryonic dorsal skin dermis, mesonephros, sclerotome, liver, and gizzard mesenchyme, whereas retina from slightly older embryos was only a weak inductor. Those lenses obtained in vitro were shown with immunohistological methods to be capable of synthesizing lens-specific proteins (Mizuno & Katoh, 1972). This synthesis is detectable even before full differentiation of the lens has taken place, which is in accordance with the development in vivo (Ikeda & Zwaan, 1967). In the present study, the lens forming capacity of the presumptive lens ectoderm of mouse and chick embryos was studied in vitro. The isolated ectoderm was grown in various culture conditions and combined with heterotopic tissues. Such permissive factors allowed lens differentiation even when the ectoderm was isolated before making contact with the optic vesicle. The inductive effect of the optic vesicle was studied by growing it in combination with the trunk ectoderm of 2-day avian embryos. In the lentoids formed, the synthesis of lens crystallins was demonstrated by immunofluorescent methods. The possibility of cell contamination was excluded by using chick-quail chimaeric combinations. MATERIALS AND METHODS Eggs of White Leghorn chicks and Japanese quail were obtained from a local poultry farm, and incubated in forced draft incubator at 37-5-38-5 C. Inbred strains A/Sn and CBA and hybrids A/CBA were used to obtain mouse embryos. Human embryonic material was obtained from early therapeutic abortions (Boije Hospital, Helsinki). Preparation and culture of the tissues All tissues were handled aseptically, rinsed and dissected in saline (mouse and human in PBS, chick and quail in Tyrode solution) prepared with disposable needles and grown on Millipore membrane niters (Millipore Corporation, Bedford, Mass.). Both agarose (LTndustrie Biologique Francaise S.A., Gennevilliers, Seine) and 0-5-2-0 % agar (Difco) made up in b (...truncated)