Camp. Biochem. Physiol.Vol.
1994 Elsevier Britain.
S6.00 + 0.00
Temporal changes in intracellular free calcium levels in the developing neuroepithelium during neurulation in the chick Joseph V. Martin, Robert G. Nagele and Hsin-yi Lee Department of Biology, Rutgers University, Camden, NJ 08102, U.S.A.; Department of Molecular Biology, University of Medicine and Dentistry of New Jersey-School of Osteopathic Medicine, Stratford NJ 08084, U.S.A. Intracellular free calcium ion (Ca*+) levels of the developing chick neuroepithelium during neural tube closure (Hamburger and Hamilton stages 3-11 of embryonic development) were determined using the hydrophobic acetoxymethyl ester of the fluorescent dye fura- (fura-Z/AM). Temporal changes in the free Ca2+ level in neuroepithelial cells are correlated with the degree of folding of the neuroepithelium. The concentration of intracellular Ca*+ in the neuroepithelium reaches its highest level when apposing neural folds are actively making contact.
Key words: Intracellular
free calcium; Neurulation.
107A, 6.55-6.59, 1994.
Introduction Neurulation in most vertebrates is a deceptively simple process in which a somewhat flattened sheet of neuroepithelial (NE) cells bends along the embryonic midline and, after a specific sequential pattern of shape changes, transforms itself into a hollow cylinder. The resulting neural tube is the rudiment of the entire central nervous system (CNS). Although neural tube closure may be caused by a complex interplay of multiple factors, apical microfilament-mediated changes in the shape of NE cells (especially apical constriction) are now thought to serve as a major source of driving forces requisite for bending of the neuroepithelium during closure of the neural tube (Nagele et al., 1989). Studies using indirect immunofluorescence have shown that apical circumferential microfilament bundles (CMBs) of developing NE cells contain motility-related proteins such as actin and myosin (Nagele and Lee, 1978, 1980; Sadler et al., 1982). Also, it is well known that the
cytoplasmic contractile elements of both muscle and non-muscle cells are regulated by local modulation of intracellular free calcium ion (Ca*+) levels (see Huxley, 1969; Campbell, 1983). The possibility therefore exists that Ca*+ regulates apical CMB contraction in the developing neuroepithelium in a manner similar to the regulation of skeletal muscle contraction. This view is supported by the finding that experimental perturbations of intracellular free Ca*+ levels alter the normal pattern of elevation and alignment of neural folds by modulating the contractile activity of apical CMBs of NE cells (e.g. Moran, 1976; Moran and Rice, 1976; Lee et al., 1978a; Lee and Nagele, 1979, 1985, 1986; O’Shea, 1982). However, there is little or no information about temporal changes in intracellular Caz+ levels in the developing neuroepithelium during closure of the neural tube. Fluorescent indicators are widely used to measure intracellular free Ca*+ levels because of their sensitivity, specificity, and ease of calibration. The acetoxymethyl ester of the fluorescent dye fura- (fura-2/AM) easily permeates into intact cells because of its lipid solubility, and the free dye is then generated intracellularly
Correspondence to: Joseph V. Martin, Department of Biology, Rutgers University, Camden, NJ 08102, U.S.A. Tel. 609-225-6142. Received 18 May 1993; accepted 25 June 1993 655
Joseph V. Martin et al.
by ubiquitous esterases (Grynkiewicz et al., 1985). In the present study, we used fura-2/AM to measure intracellular free Ca2+ levels in the developing neuroepithelium during closure of the neural tube in the chick.
Materials and Methods Fertile hens eggs were incubated at 37.5”C to obtain embryos at stages 3 +-1 1 of development (Hamburger and Hamilton, 1950). Embryos at these stages were chosen for investigation because all phases of neural tube formation are represented (Nagele et al., 1989). Neuroepithelia were isolated from embryos in cold (24°C) avian Ringer’s solution. Isolated neuroepithelia from two or more developmental stages (i.e. stages 3 +-5, stages 6-7+, stages 8 --8+, stages 9--9, stages 9+-10, and stages 10+-l 1 of development) were pooled in a microfuge tube so that a total wet weight of each pooled sample was 0.24.6 mg. Intracellular free Ca2+ levels were then determined using the hydrophobic acetoxymethyl ester of fura(fura-2/AM) (Molecular Probes) as previously described (Martin et al.. 1991) with minor modifications. Briefly, each sample of pooled neuroepithelia was dispersed in cold buffered salt solution (BSS: 136 mM NaCl, 5.6 mM KCl, 1.3 mM MgC&, 11 mM glucose, 20 mM Tris HCl, pH 7.4) by repeatedly pipetting and/or trypsinization (Lee et al., 1978b). Aliquots (0.4 ml) of the resulting dispersed cells were incubated with 10 /*M fura2/AM at 37°C for 45 min with shaking. Each incubate was diluted with an additional two volumes of warm (37°C) BSS and incubated at 37°C for 15 min to maximize hydrolysis of fura-2/AM to free dye by intracellular esterases (Rezazadeh et al., 1989). To remove unbound (excess) dye, the cell suspension was centrifuged in a microfuge (Centra-M, International Equipment Company, Needham Heights, MA) at 17,OOOg for 5 min, and the resulting pellet was resuspended in fresh BSS. This centrifugation/resuspension procedure was repeated once. Dye-loaded cell suspensions (0.1 ml) were added to 1.9 ml BSS and maintained at W”C. Immediately before use, cell suspensions were incubated at 37°C for 15 min. Fluorescence was measured at 500nm in an Aminco-Bowman Model 54-8202 spectrofluorometer (Silver Spring, MD) as in our previous study (Martin spectrum was et al., 1991). An excitation measured, and a ratio of fluorescence due to two wavelengths (R = F,,,/F,,,) was taken as a preliminary baseline measurement (non-depolarized condition). Next, 0.1 ml of 945 mM KC1 was added to each sample, bringing the final concentration of KC1 to 45 mM. A second ratio was taken at 2 min after the addition of the KC1
(depolarized condition). Under these circumstances, consistently more than 85% of the fluorescence was due to fura- within NE cells, as determined by quenching with added 40 ,uM Mn*+ (Martin et al., 1991). To obtain a measure of the minimal dye response (R,,,) in the presence of very low Ca2+, 1.8 ml of 0.5% sodium dodecylsulphate (SDS) in 4 mM ethyleneglycolbis-(oxyethylenenitrilo)tetraacetic acid (EGTA) was added to a set of tubes processed along with the experimental samples (Martin et al., 1991). To estimate the background fluorescence to be subtracted, a second set of parallel incubates containing cells without dye was used. At the end of 2-min measurement of the effects of depolarization, the non-quenching Ca2+ ionophore 4-Br A23 187 (Deber et al., 1985) was added to each experimental sample at a final concentration of 5 PM. After 3 min, a final set of readings was made (R,,,) as a measure of the maximal dye response in the presence of saturating levels of Ca2+. The concentration of Ca2+ within NE cells was calculated using a Kd for furaof 225 nM (Grynkiewicz et al., 1985). After an initial determination of the resting intracellular free Ca2+ level (non-depolarized condition), the K+ concentration was raised to 45 mM, and the effect of depolarization was monitored. Free Ca2+ level in NE cells was calculated under each of the conditions (depolarized and non-depolarized). Data were subjected to a one-way analysis of variance (ANOVA) for independent groups (Snedecor and Cochran, 1980).
Results An analysis of variance (ANOVA) showed a significant effect of stage of embryonic development on the basal level of free Ca’+ in the neuroepithelial cytoplasm of cells the (P < 0.05). Levels of intracellular Ca’+ showed developmental variations that correlated with the pattern of elevation of “le neural folds. Stage 3 +-5 embryos Induction of the neural plate is thought to be initiated in stage 3+ embryos (Fig. 1A) and completed by stage 5 of development (Fig. 1B). The neural plate in a stage 5 embryo appears as a thickened parabolic ectodermal region (Fig. 1B). The average concentration of intracellular free Ca*+ was found to be 1.3 PM (Fig. 2). Neuroepithelial (NE) cells showed an unusual sensitivity of intracellular free Ca*+ levels to the depolarizing effects of high levels of K+, increasing by 358 f 139% (mean + S.E.M.) over control levels.
Stage 6-7+ embryos
By stage 6 of development, a crescentric headfold appears at the anterior border of the future head (Fig. 1C). The flattened neural plate seen in earlier embryos has been altered by thickening and elevation of its anterolateral margins, result-
ing in the formation of neural folds. The neural folds appear as a pair of dark bands extending from the head-fold posteriorly about three-quarters of the way towards the primitive pit, where they become indistinct (Fig. 1C). The intracellular free Ca2+ level within the neuroepithelium was elevated by 50% over earlier levels at Stages
Fig. I. A series of time-lapse photographs illustrating major morphogenetic events of a chick embryo explanted at stage 4 of development using a modified News technique (Lee et al., 1990) and cultured in v&o. The yolk (y) in these photographs was detached from the blastoderm and vitelline membrane (especially during earlier hours of incubation) and floated freely in nutrient medium during handling and photographing. (A) O-hour incubation (immediately after explantation). This is a stage 4 chick embryo which was obtained by incubating a fertile hen’s egg at 37S”C for 19 hr. ao, area opaca; ap. area pellucida; ps, primitive streak. (B) IS-hr incubation (head-process stage). hp, head-process; np, neural plate. (C) 3-hr incubation (early head-fold stage). hf. head-fold; nf, neural fold. (D) 4-hr incubation (head-fold stage). (E) 6-hr incubation (2-somite stage). s, somite. (F) IO-hr incubation (5somite stage). v, vitelline vein. (G) 12-hr incubation (7-somite stage). (H) 16-hr incubation (9-somite stage). ov, optic vesicle. (I) 18-hr incubation (IO-somite stage). (J) 21-hr incubation (1 I-somite stage). x 20.
Joseph V. Martin et af
the underlying area pellucida. By this time the apposing neural folds have met along most of their length (Fig. 1G). Constriction of the bases of optic vesicles becomes more prominent than at earlier hours of incubation. The concentration of intracellular free Ca*+ falls below that of stage 3+-5 embryos (Table l), and was not changed by treatment with high K+.
Fig. 2. Developmental variation in free Ca*+ in dispersed neuroepithelial cells. The concentration of Ca*+ was measured using the fluorescent dye fura- in pooled tissue from embryos collected at the indicated developmental stages. The results are presented as mean & standard error of the mean for three separate determinations made on 2 days.
3.‘-5 (Fig. 2). Depolarization with high K+ did not significantly alter the level of Ca*+ in these cells.
Stage 8-X+ embryos
The neuroepith~lium shows great variations in the degree of folding along its length in stage 8 or 8+ embryos (Fig. 1I)). Just anterior to the primitive pit, the neuroepithelium thickens to form the neural plate. In the somite and posterior hindbrain regions, neural folds are widely separated. More anteriorly (at the level of the prospective midbrain and anterior hindbrain regions), the neural folds have curled over and are approaching each other. The total intracellular free Ca’+ level in the neuroepithelium of the cephalic region steadily increased during uplifting of the neural folds (Fig. 2). Depola~zation with high K+ caused an insignificant elevation of CaZ+ levels, by 35 + 31% over control levels. Stage 9-‘-9 embryos
The neural tube in the cephalic region rapidly expands, especially in the future forebrain (Fig. 1E). The intracellular free Ca*+ level reached its highest point (4.5 PM) by approximately stage 9 of development (Fig. 2). Stage 9+-10 embryos
Three primary brain vesicles have formed, and cranial flexure is begun. Optic vesicles are prominent and show signs of constriction at their bases (Fig. 1F). The concentration of intracellular free Ca*+ began to decline (Fig. 2) in cells from these embryos. Treatment with elevated K+ did not significantly alter the concentration of free Ca2+. Stage 10+-l 1 embryos
Flexure of the crania1 region continues, and the developing brain is distinctly separated from
A substantial body of biochemical, cell biological and pharmacological evidence supports a view that Ca’+ plays a pivotal role in a variety of cellular processes. Ca*+ is likely to serve as the regulator of the contractile activity of apical circumferential microfilament bundles (CMBs) in neuroepithelial (NE) cells of the developing neuroepithelium. Experimental evidence in favour of this idea includes the fact that chemical agents (e.g. papaverine, ionophore A23 187, verapamil and local anesthetics), which are known to alter intracellular Ca2+ levels, disrupt apical constriction of NE cells and elevation of neural folds in amphibian, chick and mouse embryos (Moran, 1976; Moran and Rice, 1976; Lee et al., 1978a; Lee and Nagele, 1979, 1985, 1986; O’Shea, 1982). For Ca*+ to serve as a regulatory ion, NE cells must have a means for controlling local intracellular Ca*+ levels. We have investigated the involvement of Cat+ in neural tube closure by studying its ultrastructural localization in the developing neuroepithelium (Bush et al., 1992). Ca2+ is found most prominently in lipid droplets situated at the apical ends of NE cells. Although the physiological significance of high Ca2+ con~ntrations in apical lipid droplets of NE cells remains unclear, these lipid droplets may serve to store and slowly release Ca2+ and thereby regulate the slow, but progressive, constriction of the apical ends of NE cells during closure of neural folds. Displacement of Ca2+ from the culture medium is known to inhibit elevation of neural folds (Smedley and Stanisstreet, 1985; Moore and Stanisstreet, 1986) whereas addition of Ca2+ to embryos with collapsed neural folds causes the neural folds to elevate again (O’Shea, 1982). Our recent study has shown that transient elevation of free Ca” levels by ionomycin dramatically accelerates the rate of neural tube formation (Lynch er al., 1989). The rapidity of the response (hours of normal development reduced to a few minutes) along with the increased prominence of apical CMBs and the degree of apical constriction of NE cells strongly supports a direct causal role for apical CMBs of NE cells in bending of the neuroepithelium. Furthermore, the extremely short time frame of these developmental accomplishments effectively rules out the possibility
levels durir Ig chick
that changes in the extracellular matrix, development pressures from surrounding structures and local increases in the mitotic index make major contributions as sources of extrinsic force for closure of the neural tube. The present study shows that, as neural folds are actively uplifted during stages 6-8 of embryonic development, there is a steady increase in the intracellular free Ca*+ level in the neuroepithelium (Fig. 2). In the earliest examined embryonic tissue (stages 3+-5), there are also indications of voltage sensitivity of the tissue in increasing Ca*+ permeability or mobilization of Ca*+ from internal stores. At or near stage 9 of development, when apposing neural folds are actively making contact, intracellular free Ca*+ in the neuroepithelium reaches its highest level. Once the apposing neural folds have met along most of their length (e.g. in stage 10 embryos), the concentration of intracellular free Ca*+ begins to decrease. These correlations would be expected if Ca*+ were indeed serving as a regulatory ion of apical constriction of NE cells. More specifically, since the level of intracellular free Ca2+ increases while the neural folds are actively being uplifted, these data are in accord with the hypothesis that Ca*+ regulates changes in the shape of NE cells by controlling the contractile activity of their apical CMBs. Atknowledgements-We thank Donald R. Keir and Matthew W. McConville for their technical assistance. This study was supported by grants from the National Institutes of Health (NS23200) and the Busch Fund of Rutgers Ilniversity.
References Bush K. T., Lee H. and Nagele R. G. (1992) Lipid droplets of neuroepithelial cells are a main calcium storage site during neural tube formation in chick and mouse embryos. Experienria 48, 5 16-518. Campbell A. K. (1983) Intracellular calcium: Its Universal Role as a Regulator. Wiley, New York. Deber C. M., Tom-Jun J., Mack E. and Grinstein S. (1985) Bromo-A23187: A nonfluorescent calcium ionophore for use with fluorescent probes. Anal. Biochem. 146, 349. Grynkiewicz G., Poenie M. and Tsien R. Y. (1985) A new generation of Ca ‘+ indicators with greatly improved fluorescence properties. J. biol. Chem. 260, 3440-3450. Hamburger V. and Hamilton H. L. (1950) A series of normal stages in the development of the chick embryo. J. Morphol. 88, 49-92. Huxley H. E. (1969) The mechanism of muscular contraction. Science 34, 1356-1366. Lee H., Nagele R. G. and Karasanyi N. (1978a) Inhibition
of neural tube closure by ionophore A23187 in chick embryos. Experientia 34, 5 18--S 19. Lee H., Kalmus G. W. and Nagele R. G. (1978b) Studies on dispersed unincubated chick blastoderm cells. II. Formation of contacts and cell sorting in aggregates of unincubated chick blastoderm and heart cells. Poultry Sci. 51, 1755-1960. Lee H. and Nagele R. G. (1979) Neural tube closure defects caused by papaverine in explanted early chick embryos. Teratology 20, 321-332. Lee H. and Nagele R. G. (I 985) Neural tube defects caused by local anesthetics in early chick embryos. Teratology 31, 119-127. Lee H. and Nagele R. G. (1986) Toxic and teratologic effects of verapamil on early chick embryos: Evidence for the involvement of calcium in neural tube closure. Teratology 33, 203-2 I I. Lynch F. J., Bush K. T., McConville M. W., Lee H. and Nagele R. G. (1989) Acceleration of neural tube formation in chick and mouse embryos treated with ionomycin: Evidence of a regulatory role of calcium. J. Ceil Biol. 109, 63a. Martin J. V., Keir D. R. and Lee H. (1991) Diazepam enhances intrasynaptosomal free calcium ion concentration. Brain Res. 548, 222-227. Moore D. C. P. and Stanisstreet M. (1986) Calcium requirement for neural fold elevation in rat embryos. Cyrobios 41, 167-177. Moran D. (1976) Scanning electron microscopic and flame spectrometric study on the role of Ca’+ in amphibian neurulation using papaverine inhibition and ionophore induction of morphogenetic movements. J. esp. Zoo/. 198, 409416. Moran D. and Rice R. W. (1976) Action of papaverine and ionophore A23187 on neurulation. Nature, Land. 261, 497499. Nagele R. G. and Lee H. (1978) Motility-related proteins in developing neural cells. Am. Zoo/. 18, 608. Nagele R. G. and Lee H. (1980) Studies on the mechanisms of neurulation in the chick: microfilament-mediated changes in cell shape during uplifting of neural folds. J. exp. Zool. 213, 391-398. Nagele R. G., Bush K. T., Hunter E. T., Kosciuk M. C.. Steinberg A. B. and Lee H. (1989) Intrinsic and extrinsic forces collaborate to generate driving forces for neural tube formation in the chick: a study using morphometry and computerized three-dimensional reconstruction. Dev. Brain Res. 50, IOI--III. O’Shea K. S. (1982) Calcium and neural tube closure defects: an in vitro study. Birth D+crs: Original Article Series 18, 95-106. Sadler T. W., Greenberg D.. Coughlin P. and Lessard J. L. (1982) Actin distribution patterns in the mouse neural tube during neurulation. Science, Wash. 215, 172-174. Rezazadeh M., Woodward J. J. and Leslie S. W. (1989) Furameasurement of cytosolic free calcium in rat brain cortical synaptosomes and the influence of ethanol. Alcohol 6, 341-345. Smedley M. J. and Stanisstreet M. (1985) Calcium and neurulation in mammalian embryos. J. Embryo/. esp. Morphol. 89, I-14. Snedecor G. W. and Cochran W. G. (1980) Sratisrical Methods, p. 215. The Iowa State University Press, Ames, Iowa.