Reliability of delta-crystallin as a marker for studies of chick lens induction

Induction of a lens by the optic vesicle of the brain was the first demonst ration of how tissue interactions could influence cell fate during developm ent. However, recent work with amphibians has shown that the optic vesicle is not the primary inducer of lens formation. Rather, an earlier interactio n between anterior neural plate and presumptive lens ectoderm appears to di rect lens formation. One problem with many early experiments was the absenc e of an unambiguous assay for lens formation. Before being able to test whe ther the revised model of lens induction applies to chicken embryos, we exa mined the suitability of using delta-crystallin as a marker of lens formati on. Although delta-crystallin is the major protein synthesized in the chick lens, one or both of the two delta-crystallin genes found in chickens is t ranscribed in many non-lens tissues as well. In studies of lens formation w here appearance of the delta-crystallin protein is used as a positive assay , synthesis of delta-crystallin outside of the lens could make experiments difficult to interpret. Therefore, polyacrylamide gel electrophoresis, immu noblotting, and immunofluorescence were used to determine whether the delta -crystallin messenger RNA detected in non-lens tissues is translated into p rotein, as it is in the lens. On Coomassie-blue-stained gels of several tis sues from stage-22 embryos, a prominent protein was observed that co-migrat ed with delta-crystallin. However, on immunoblots, none of the nonlens tiss ues tested contained detectable levels of delta-crystallin at this stage. B y imunofluorescence, deltacrystallin was observed in Rathke's pouch and in a large area of oral ectoderm near Rathke's pouch, yet none of the cells in these non-lens tissues showed the typical elongated morphology of lens fib er cells. When presumptive lens ectoderm or other regions of ectoderm from stage-10 embryos were cultured and tested for lens differentiation, both ce ll elongation and delta-crystallin synthesis were observed, or neither were observed. The results suggest that delta-crystallin synthesis and cell elo ngation together serve as useful criteria for assessing a positive lens res ponse.