| A requisite step in assessing the role of cytoplasmic contractile proteins in providing the force necessary for optic cup formation is the determination of Ca('2+)-dependence for this morphogenetic process. Stage 12-13 chick embryo heads were excised and cultured overnight in nutrient medium containing agonists or antagonists of Ca('2+) transport. The antagonists of Ca('2+) transport, papaverine and verapamil, reversibly inhibited optic cup formation compared to control heads which were cultured in the absence of any drugs. The Ca('2+) ionophore A23187 initiated precocious optic cup formation (within 30 min after exposure) only in the presence of external Ca('2+). In the absence of external Ca('2+) A23187 did not initiate precocious optic cup formation. Neither theophylline nor caffeine, mobilizers of intracellular Ca('2+), were able to initiate precocious cup formation. When heads were excised and incubated overnight in the presence of the calmodulin inhibitors trifluoperazine (TFP) and chlorpromazine (CPZ) optic cup formation was reversibly inhibited compared to untreated controls. The concentrations of TFP and CPZ needed to inhibit optic cup formation directly correlate with the relative concentrations of these compounds necessary for the inhibition of calmodulin in vitro. Chlorpromazine sulfoxide, which is ineffective at binding and inactivating calmodulin in vitro, had no inhibitory effect on optic cup formation at the concentration utilized (10 times the inhibitory concentration of CPZ). Indirect immunofluorescence studies indicated that calmodulin is homogeneously distributed throughout the cells of the optic vesicle. The binding of ('3)H-TFP to intact stage 13 heads indicated a class of high affinity, low capacity, Ca('2+)-dependent binding sites for the drug. These binding sites have a Kd of approximately 5 (mu)M which correlates directly with the Kd for pure calmodulin in vitro (Kd = 1 (mu)M). These data suggest that optic cup formation is a Ca('2+)-dependent process. The source of the Ca('2+) seems to be extracellular. Calmodulin probably confers the Ca('2+)-dependence to this system. These data are consistent with calmodulin functioning to regulate optic cup formation by mediating actin-myosin interaction via phosphorylation of the 20,000 dalton light chain of myosin. |
