Insulin improves survival but does not maintain function of cultured chick wing bud apical ectodermal ridge

Previously we demonstrated that high levels of insulin (5 micrograms/ml) permit the survival of isolated chick apical ectodermal ridge in culture (Boutin and Fallon, Dev. Biol., 104:111-116, 1984). Here we address whether lower levels of insulin or insulin-like growth factors (IGFs) can also improve the survival of cultured apical ectodermal ridge and whether ridge function is maintained along with ridge survival. Neither IGF I nor IGF II (100 ng/ml) decreased ridge cell death; however, cell death was significantly decreased with 50 ng/ml insulin. No further improvement was obtained in the presence of both IGF I and insulin. These data suggest that insulin improved the survival of the isolated apical ectodermal ridge by binding its own receptor. To test for the maintenance of function, stage 20 ridges were cultured for 0, 6, 12, 18, or 24 hr with or without insulin (5 micrograms/ml or 5 ng/ml) and used to make recombinant limbs. Isolated ridges cultured for 12 hr or more produced fewer outgrowths and these were rarely distally complete. The medium in which the ridges had been cultured did not influence ridge activity, despite the major differences in cell survival. Recombinants made with ridges cultured with limb mesoderm for 18 hr did not yield outgrowths as often as those with freshly isolated ridges, but most of the limbs that did form were distally complete. These results suggest that the decline in function of cultured, isolated apical ectodermal ridge was not due merely to ridge cell death but rather, at least in part, to its separation from limb mesoderm.