Optic cup formation: a calcium-regulated process.

Abstract

Invagination of the optic vesicle to form the optic cup is an important event in the formation of the eye in the early embryo. To obtain support for earlier conclusions that a contractile process is involved, calcium dependency ofoptic cup formation was tested. Heads were excised from chicken embryos at the optic vesicle stage of development (stage 13) and incubated in nutrient medium containing antagonists or agonists of calcium transport. Invagination was reversibly inhibited by the Ca2+ antagonists verapamil and papaverine. It was initiated in a precocious fashion by the Ca2+ ionophore A23187 but only in the presence of external Ca2+. Neither caffeine, theophylline, nor A23187 (in the absence of external Ca2+) were able to initiate precocious optic cup formation. Trifluoperazine and chlorpromazine reversibly inhibited optic cup formation while chlorpromazine sulfoxide had no effect at the concentrations used. Reagents. The calcium ionophore A23187 was purchased from Calbiochem-Behring and was dissolved in dimethyl sulfoxide. Theophylline, papaverine, and caffeine were purchased from Sigma. Verapamil was supplied by Knoll Pharmaceutical (Whippany, NJ). Trifluoperazine (TFP), [3H]TFP (67 ,uCi/mg; 1 Ci = 3.7 X 10O' becquerels), chlorpromazine (CPZ), and chlorpromazine sulfoxide (CPZ-S02) were the gifts of Smith Kline & French.Antisera. Sheep anticalmodulin (rat testis) was the gift of John Dedman (University of Texas Medical School). Fluorescein-conjugated rabbit anti-sheep IgG was purchased from GIBCO.Culture Experiments. All cultures were incubated at 37°C in medium 199 (GIBCO) in humid 5% C02/95% air. The inhibitory effects of various drugs were studied in an overnight culture system. The heads of stage 12 to 13 chicken embryos were excised, placed in medium containing a variety of concentrations of calcium antagonists or phenothiazines, and then incubated for 18 hr. The phenothiazines and papaverine were dissolved in water and diluted to the appropriate concentrations with medium. Verapamil was dissolved in 95% ethanol. Control heads were placed in medium containing no drug and incubated in parallel with each group of experimental heads. After 18 hr, experimental heads were examined by light microscopy and compared with controls for optic cup formation. Photographs were taken through a Wild M5 (Wild-Heerbrugg, Farmingdale, NY) dissecting microscope. Precocious optic cup formation in response to calcium agonists was assessed in a short-term (30-min) culture system.[3H]TFP Binding Studies. Heads of stage 13 chicken embryos were excised with extreme care to ensure equal size. They were incubated at 37°C in the presence of various concentrations of [3H]TFP (67 ,Ci/mg) for various periods of time as stated in the figures. After incubation, each head was individually transferred through three successive wash vials containing 10 ml of ice-cold medium. The medium used for washing was always the same as that used in the initial incubation except that it contained no drug. Each head was kept in each wash vi...

Second messenger regulation of occlusion of the spinal neurocoel in the chick embryo

“…This interpretation is further supported by the finding that none of 48 stage 11-13 + embryos exhibited reopening of the occluded neurocoels when treated for 4 h r in medium 199 with agents known to mobilize intracellular Ca2+: 10 mM caffeine or theophylline. The concentration of these two drugs was identical to that used by Brady and Hilfer (1982) to show that in chick embryos of stages similar to those used in our study, optic cup morphogenesis was not reversed by 10 mM caffeine or theophylline.…”

“…To determine whether occlusion requires calmodulin, embryos with occluded neurocoels were treated with three pharmacological agents known to be antagonists of calmodulin: chlorpromazine (CPZ) (Brady and Hilfer, 1982;Levin and Weiss, 1979), trifluperazine (TFP) (Levin and Weiss, 19771, and N-(6-aminohexyl)-5-chloro-1-napthalenesulfonamide (W-7) (Hidaka et al, 1981). The rationale for these treatments was based on the prevailing cell biology theory that all calmodulin reactions require Ca2' (Cheung, 1980).…”

“…It could also be argued that Ca2+ mediated its effect on neurocoel occlusion via a putative intrinsic mechanism: contraction of cytoplasmic actomyosin, considered to be a major force in bending the neural plate into a tube Nagele, 1985, 1988;Moran and Rice, 1976) and the invagination of the optic vesicle to form the optic cup (Brady and Hilfer, 1982). Furthermore, it is well known that actomyosin interaction in nonmuscle cells is a process dependent on Ca2+ (Brady and Hilfer, 1982;Cohen and de Vries, 1973;Korn, 1978) and calmodulin (Cheung, 1980;Yerna et al, 1979). However, when Desmond and Schoenwolf (1986) treated embryos with cytochalasin D a t concentrations effective in depolymerizing microfilaments, the neurocoels remained occluded.…”

Basal lamina heparan sulphate proteoglycan is involved in otic placode invagination in chick embryos

“…It has been postulated that the cellular cytoskeleton plays a role in the folding of numerous embryonal epithelial primordia (Hilfer and Searls 1986), and changes in the organisation of actin and myosin could bring about constriction of cell apices, inducing epithelial invagination. In this regard experimental manipulation of ATP levels or disruption in the transport of Ca ++ , conditions that interfere with the interaction of cytoskeletal actin and myosin, have been shown to disrupt invagination of the thyroid placode (Hilfer et al 1977), optic vesicle (Brady and Hilfer 1982), nasal placode (Smuts 1981) and neural plate (Lee et al 1983;Schoenwolf et al 1988;Ferreira and Hilfer 1993). Nevertheless, these manipulations apparently have no effect on invagination of the otic placode (Hilfer et al 1989), which indicates that the factors in question do not play a decisive role in the epithelial folds leading to otic vesicle formation.…”

Chondroitin Sulphate Proteoglycan is Involved in Lens Vesicle Morphogenesis in Chick Embryos

“…It has been suggested that this mechanism operates during the invagination of different ectodermic placodes such as the lens itself, the otic and nasal placodes and the neural tube, among others (Wrenn and Wessells, 1969;Bancroft and Bellairs, 1977;Brady and Hilfer, 1982;Ferreira and Hilfer, 1993); (2) a high rate of cell replication in the lens placode, with respect to the surrounding surface ectoderm, has been proposed as the mechanism responsible for creating forces involved in lens placode invagination (Modak, Morris and Yamada, 1968;Harding et al, 1971;Hendrix and Zwaan, 1974;A Â lvarez and Navascues, 1990;Hendrix, Madras and Johnson, 1993); (3) certain extracellular factors and, more speci®cally, extracellular matrix molecules, appear to be associated with epithelial invagination. What is more, it is known that alteration of the synthesis or enzyme degrading of particular matrix molecules such as collagen, certain proteoglycans and glycoproteins, may disrupt epithelial folding, as occurs in the branching of the salivary gland, lung and kidney primordia (Spooner and Faubion, 1980;Thompson and Spooner, 1983;Spooner, Bassett and Stokes, 1985;Nakanishi et al, 1986;Klein et al, 1989).…”