combination of endoderm and mesoderm, the splanch- nopleure, and is connected to the midgut of the embryo by an open tube, the yolk duct, so that the walls ...
Conference: Molecular Aspects of Vitamin and Mineral Nutrition during Avian Development
Developmental Expression and Vitamin D Regulation of Calbindin-D28K in Chick Embryonic Yolk Sac Endoderm1'2 ROCKY S. TUAN3 AND E1KO SUYAMA Department of Orthopaedic Surgery, Thomas Jefferson Uniuersity, Philadelphia,
present in the yolk sac. We report here the develop mental expression and regulation of calbindin-D28K in the yolk sac. Calbindin-D2aKis expressed as early as incubation d 3 and is found exclusively within the cyto plasm of endoderma! cells. Comparative protein and mRNA analyses of yolk sac and dissociated yolk sac endoderma! cells as a function of development and treatment with calcitrici ( 1,25-dihydroxy vita min D3) in vitro and in vivo showed a development-specific and vitamin D-inducible expression of calbindin-D28K• Northern analysis revealed the expression of vitamin D receptor mRNA in the yolk sac, beginning as early as d 3, strongly indicating that the extraembryonic yolk sac is an early vitamin D target tissue. Cultured yolk sac endoderma! cells should serve as a useful in vitro cell model for analyzing the cellular and molecular mechanisms of vitamin D action. J. Nutr. 126: 1308S-1316S, 1996.
Henry and Norman 1978, Sunde et al. 1978, Narbaitz and Tsang 1989, Narbaitz et al. 1987). Such vitamin Ddeficient chick embryos consistently showed very low hatchability and exhibited hvpocalcemia, hyperphosphatemia and undermineralized skeleton. During development, the chick embryo acquires the necessary calcium from two extraembryonic sources, the yolk and the eggshell (Johnston and Comar 1955). Until about incubation d 10, the yolk acts as the only calcium source,- thereafter, calcium is also mobilized from the eggshell (see reviews by Packard and Packard 1984, Tuan 1987, Tuan et al. 1991). Building on the initial observations that chick embryos cultured ex ovo in the absence of the eggshell were capable of a hyper calcémieresponse to calci triol (Narbaitz 1979, Tuan and Ono 1986), we and others subsequently demon strated that the process of yolk calcium mobilization is indeed regulated by calcitriol and that the yolk sac, which translocates yolk calcium, is a major target tis sue of the hormonal action of vitamin D on embryonic calcium homeostasis (Clark et al. 1989, Lee and Clark 1993, Lee et al. 1990, Ono and Tuan 1991 ). Specifically, calcitriol treatment of chick embryos enhanced cal-
INDEXING KEY WORDS:
•calbindin •yolk sac •vitamin D •calcium transport •epithelial transport
A number of studies have clearly demonstrated the importance and requirement of vitamin D in chick em bryonic development. These investigations ranged from the classical studies that demonstrated a sharp decrease in hatchability in embryos from vitamin Ddeficient hens (Bethke et al. 1936, Branion and Smith 1932) to more recent studies in which vitamin D-deficient embryos were generated from the feeding of lay ing hens with 1,25-dihydroxy vitamin D3 (calcitrici) substituting for vitamin D (Hart and DeLuca 1985,
' Presented as part of the 60th Annual Poultry Nutrition Confer ence: "Molecular Aspects of Vitamin and Mineral Nutrition during Avian Development" given at the Experimental Biology '95 meeting, Atlanta, GA, on April 9, 1995. This conference was sponsored by the American Institute of Nutrition and was supported in part by grants from Archer Daniels Midland Company, Merck & Company Inc., Pilgrim's Pride Corporation, Prince Agri Products Inc., Shaver Poultry Breeding Farms, and U.S. Department of Agriculture, Ag ricultural Research Service. The guest editor for the conference publi cation was Mark P. Richards, Growth Biology Laboratory, U.S. De partment of Agriculture, Agricultural Research Service, Beltsville, MD. 2 Supported in part by grants from the NM (HD 15822, HD 29937 and ES 07005). 3To whom correspondence should be addressed: Department of Orthopaedic Surgery, Thomas Jefferson University, 1015 Walnut Street, Philadelphia, PA 19107.
0022-3166/96 $3.00 ©1996 American Institute of Nutrition.
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ABSTRACT The yolk is an important calcium source for the developing chick embryo. The epithelial yolk sac endoderma! cells lie in direct contact with the yolk and are the principal nutrient-transporting cell type. We previously reported that vitamin D treatment stim ulated yolk calcium mobilization andcalb¡nd¡n-D¿RK, that the vitamin D-dependent Ca' -binding protein, is
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mercial egg incubator. After 3 d of incubation in ovo, embryonated eggs were cracked open aseptically and transferred into a hemispherical pouch made of trans parent plastic kitchen wrap suspended within a ring stand. The culture was loosely covered with a 100-mm Petri dish lid and then placed in a humidified tissue culture incubator at 37.5°C with constant air flow (Tuan 1980). At total incubation time of 3 d, 6 d and 9 d, each embryo received 50 ¡jiL of 95% ethanol either with or without 300 pmol of calcitriol (Biomol, Plym outh Meeting, PA) injected into the egg yolk (Ono and Tuan 1991, Tuan and Ono 1986) and incubated further for 2 or 3 ds.
FIGURE 1 Diagrammatic representation of the processing and analysis of chick embryonic yolk sac specimens. Details are provided in the text in Materials and Methods. SL, shellless culture of whole embryos; vit. D, calcitriol (300 pmol per embryo in ovo, 150 nmol/L for organ culture or 1 nmol/ L for endodermal cell culture).
MATERIALS AND METHODS Shell-less culture of chick embryos ex ovo. Fertile White Leghorn eggs obtained from Truslow Farms
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FIGURE 2 Expression of calbindin-D2sK in d 3 chick em bryonic yolk sac detected by immunohistochemistry. Yolk sacs from embryos incubated for 3 d in ovo were sectioned and immunostained as described in Materials and Methods and Figure la and b. Positive calbindin-D28K staining cells are seen (arrows) and are more abundant in the area vasculosa (b) than the area vitellina (a). Calbindin-D28K is localized to the cytoplasm of the endoderm cells, which are characterized by the presence of vacuolated regions representing internal ized yolk granules (*). (c) higher magnification of (b). (d) con trol omitting calbindin-D28K antibody, showing absence of staining. Bar = 50 ¿im(a, b and d} or 25 /¿m(c).
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cium uptake by the yolk sac as measured in vivo (Tuan and Ono 1986) and in vitro (Lee and Clark 1993, Lee et al. 1990). Interestingly, the vitamin D-dependent Ca2+binding protein (calbindin-D2gK) is detected in the yolk sac (Ono and Tuan 1991, Sechman et al. 1994); further more, the expression of calbindin-D28K is up-regulated by calcitriol (Ono and Tuan 1991). These findings strongly suggest that the hormonal action of calcitriol on yolk sac calcium transport is mediated by the regu lated expression and activity of calbindin-D28K/ analo gous to the response of the adult intestine (Feher et al. 1992, Wasserman and Fullmer 1989). The yolk sac consists of a highly vacuolated and vascularized columnar epithelium formed from the combination of endoderm and mesoderm, the splanchnopleure, and is connected to the midgut of the embryo by an open tube, the yolk duct, so that the walls of the yolk sac and the walls of the gut are continuous (Juurlink and Gibson 1973, Lambson 1970). Transport of nutrients by the yolk sac is generally believed to be of an endocytic nature involving the intemalization of yolk droplets (Mobbs and McMillan 1981). The mecha nism of calcium transport by the yolk sac probably involves endocytosis as well as other unknown mecha nisms (Komazaki et al. 1993, Lee and Clark 1993). To further examine the importance and role of vita min D in chick embryonic development, we examined in detail the ontogeny of the vitamin D responsiveness of the chick embryonic yolk sac epithelium on the ba sis of the regulated expression of calbindin-D2gK. Our results indicate that the developing yolk sac exhibits vitamin D responsiveness as early as d 3 of incubation, suggesting that vitamin D may be important for early embryogenesis.
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The dissociated cells were plated in 100-mm tissue culture dish (Falcon-Becton Dickinson Labware, Lin coln Park, NJ) and cultured in fresh DMEM medium as described for the organ cultures. Calcitriol (1 nmol/ L) was added to the cultures either immediately (short-term cultures) or after 7 d of incubation (longterm cultures), followed by two additional days of in cubation. Immunohistochemistry. Tissues were fixed in mod ified Camoy's solution for 3 h at -20°C, embedded in
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\ FIGURE 3 Vitamin D responsiveness of calbindin-D28K gene expression in chick embryonic yolk sac at d 3 and d 6 of incubation detected by immunohistochemistry. Chick embryos were maintained in shell-less culture and injected on d 3 or d 6 with or without calcitrici, allowed to develop for two additional days, and the yolk sac examined immunohistochemically for calbindin-D28K (see Materials and Meth ods and Fig. 1). D 3 (total d 5) control (a) and vitamin Dtreated (b) yolk sac, clearly showing profuse induction of calbindin-D2KK expression by vitamin D treatment. D 6 (total d 8) control jc) and vitamin D-treated (d} yolk sac, again showing stimulation of calbindin-D2SK expression by vitamin D, although the level is significantly less than the younger, d 3 yolk sac. Bar = 50 /mi.
All animal protocols were approved by the Institu tional Animal Use and Care Committee of Thomas Jefferson University. Yolk sac organ culture. Yolk sac tissue was dis sected out aseptically and cultured in Dulbecco's modi fied Eagle's medium (DMEM; Sigma, St. Louis, MO) containing 10% heat inactivated fetal bovine serum (Hyclone, Logan, UT), 200 units of penicillin and strep tomycin (Sigma), either with or without 150 nmol/L of calcitriol for 1 d at 37°C,5% CO2 in a humidified tissue culture incubation. Yolk sac endoderma! cells. Yolk sac tissue from various developmental stages was dissected out asep tically and dissociated in DMEM medium by brief vortexing using the procedure of Young and Klein (1983).
ing was done as described previously (Ono and Tuan 1991) by using antibodies directed against calbindinD28K (Taylor and Wasserman 1970) generated and kindly provided by Robert H. Wasserman (Cornell Uni versity, Ithaca, NY). A commercial, streptavidin-biotin based kit (Zymed, San Francisco, CA) was used for immunodetection according to the manufacturer's in structions. The rabbit-derived antibodies to chick calbindin-D28K were used at 1:1000 dilution. Western immunoblot. Yolk sac tissue was homoge nized in 5 mmol/L Tris-HCl, pH 7.4, and the soluble extract was obtained by centrifugation at 31,000 X g for 30 min (Ono and Tuan 1991). Protein samples were loaded at 12 /zg per lane on a sodium dodecyl sulfate 12% polyacrylamide gel (SDS-PAGE) for electrophoretic separation. The gel was subsequently electroblotted onto nitrocellulose and calbindin-D28K immunodetected using antibodies to chick calbindin-D28K at a di lution of 1:600 as described previously (Ono and Tuan 1990 and 1991). Northern UNA blot. Total cellular RNA was iso lated from yolk sac tissue using guanidine thiocyanate (Chomczynski and Sacchi 1987). Ten micrograms of total RNA was loaded on a 1% agarose gel containing formaldehyde and transferred onto GeneScreen Plus membrane (NEN-DuPont, Boston, MA). The cDNA probes used were as follows: 1 ) chicken calbindin-D28K cDNA (Mangelsdorf et al. 1987), kindly provided by Barry Komm; and 2} avian vitamin D receptor cDNA (Meyer et al. 1992), kindly provided by Mark Haussler. The probes were 32P-labeled using commercial kits for random priming (MegaPrime, Amersham, Arlington Heights, IL; for calbindin-D28K and /0-actin) or riboprobing (Riboprobe System, Promega, Madison, WI; for vita min D receptor). Hybridization was done in buffer con taining 1% SDS, 0.5 M NaCl, 10% dextran sulfate over night at 60° or 70°C, respectively, as described previously (Sato and Tuan 1992). The blots were also rehybridized with /0-actin probe to normalize calbindin-D28K and vitamin D receptor mRNA levels with respect to total mRNA content. A schematic diagram depicting the preparation and processing of the yolk sac for the various analysis de scribed above is shown in Figure 1.
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paraffin and sectioned. Isolated cells were fixed in 4% paraformaldehyde in phosphate buffered saline (PBS), pH 8.0 for 15 min at 4°C.Immunohistochemical stain
CALBINDIN IN EMBRYONIC YOLK SAC
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b). The immunoreactive signal is cytoplasmic and is distributed evenly in the nonvacuolated regions of the endodermal cell (Fig. 2c). The specificity of the calbindin-D28K immunostaining was demonstrated by the lack of signal in the control that lacked primary anti bodies (Fig. 2d). We next investigated whether this early expression of calbindin-D28K in the developing yolk sac is constitu tive or dependent on vitamin D. For this purpose, the embryonated egg was maintained in shell-less culture to facilitate administration of calcitriol, and calbindinD28Kexpression in the yolk sac was observed as a result of calcitriol treatment. As shown in Figure 3a and b,
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FIGURE 4 Vitamin D responsiveness of calbindin-D28K gene expression in chick embryonic yolk sac at d 6 and d 9 of incubation detected by immunohistochemistry. (a and b) expression of calbindin-D28K in d 6 (a )and d 9 (b ) yolk sac from embryos developing in ovo. The d 9 yolk sac shows a generally higher level of calbindin-D28K expression, (c and d)d9 yolk sac placed into organ culture in vitro and treated with calcitrici showed highly elevated level of calbindin-DMK expression in all endodermal cells (c, low magnification; d, high magnifica tion), (e and /) d 9 shell-less chick embryo injected with calcitriol on d 8 (/) showed significantly increased level of calbindin-D28K expression, compared with untreated control (e}. Bar = 50 ¿trn[a, b, c and e} or 25 pm {d}.
RESULTS During early chick embryogenesis, the yolk sac forms by progressive outgrowth of the endoderm over the yolk beneath the superficial layer of ectoderm (Ro manoff 1960). The first sign of specialization of the early yolk sac is the formation of the concentric zones: the medial, vascularized region known as area vascu losa, and the peripheral, less vascular region termed the area vitellina. We were interested in assessing the ontogeny and cellular localization of calbindin-D28K ex pression in the yolk sac. As shown in Figure 2, calbindin-D28K expression was clearly detectable in the yolk sac of the d 3 chick embryo. Immunopositive cells were seen in both areas of the yolk sac, with more being detected in the area vasculosa (compare Fig. 2a and
FIGURE 5 Immunohistochemical analysis of vitamin D stimulation of calbindin-D28K expression in cultured yolk sac endodermal cells from d 3 yolk sac. Endodermal cells were isolated from d 3 yolk sac, plated into culture and subjected to treatment with or without calcitriol for 2 d either immedi ately (short-term culture) or after 7 d (long-term culture) in culture (see Materials and Methods and Fig. 1). [a and b) short-term control (a) and vitamin D-treated (b) cultures, showing a moderate level of stimulation of calbindin-D28K expression by vitamin D. (c and d) long-term control (c) and vitamin D-treated (d ) cultures, showing distinct maturation and hypertrophy of the endodermal cells as a function of culture time and the substantial stimulation of calbindinD28Kexpression by vitamin D. Note the large cell size and the even staining throughout the cytoplasm, except the pe ripheral edge of the cell, (e) long-term cultures stained with out calbindin-D28K antibody showing the absence of staining. Bar = 50 fan.
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tion. To further examine this at a cellular level, the yolk sac from embryos at various developmental ages was nonenzymatically dissociated into single cells us ing the protocol of Young and Klein (1983). The isolated cells were placed in culture and treated for 2 d with calcitriol either immediately (short-term culture) or after 7 d in culture (long-term culture). The results for yolk sac endodermal cells isolated from d 3 embryos are shown in Figure 5. The isolated cells consisted mainly of endodermal cells, characterized by their re tention of yolk droplets in the cytoplasm. Calcitriol stimulated calbindin-D28K expression moderately in short-term culture (Fig. 5a and b). After a 7-d culture period, the endodermal cells underwent significant maturation and hypertrophy (Fig. 5c and d); interest ingly, calcitriol treatment resulted in a high level of calbindin-D28K expression in these large, hypertrophie
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FIGURE 6 Immunohistochemical analysis of vitamin D stimulation of calbindin-D28K expression in cultured yolk sac endodermal cells isolated from d 6 yolk sac. The experimen tal design is similar to that in Figure 5. (a and b] short-term control (a) and vitamin D-treated (b) cultures, showing a sig nificant level of stimulation of calbindin-D28K expression by vitamin D. Note that compared with d 3 endodermal cells, the d 6 cells showed distinct increased internalization of yolk droplets and that in the untreated cells, calbindin-D28K level is minimal, (c and d) long-term control (c) and vitamin Dtreated (d} cultures, showing distinct maturation and hyper trophy of the endodermal cells as a function of culture time and the substantial stimulation of calbindin-D28K expression by vitamin D. Bar = 50 p.m.
calcitriol injection at d 3 of incubation resulted 2 d later in a profuse expression of calbindin-D2gK com pared with control. Interestingly, although calcitriol treatment at d 6 of incubation also appeared to stimu late yolk sac calbindin-D28K expression, the relative magnitude of stimulation was less than that observed for the d 3 embryo (Fig. 3c and d). The developmental profiles of calbindin-D28K expression and of its regula tion by calcitriol were further studied in the yolk sac of d 9 embryos (Fig. 4). The d 9 yolk sac showed a generally higher level of calbindin-D28K expression compared with the d 6 yolk sac (Fig. 4a and b) and was highly stimulated by calcitriol treatment either in vitro (Fig. 4c and d) or in vivo (Fig. 4e and f ). The specific expression of calbindin-D28K in the en dodermal epithelial cells of the yolk sac strongly sug gested that these cells are the target of vitamin D ac
FIGURE 7 Immunohistochemical analysis of vitamin D stimulation of calbindin-D28K expression in cultured yolk sac endodermal cells isolated from d 9 yolk sac. Experimental design is similar to that in Figures 5 and 6. (a and b) shortterm control (a) and vitamin D-treated (b) cultures, snowing a significant level of stimulation of calbindin-D28K expression by vitamin D. Note that the d 9 cells showed the highest level of internalized yolk droplets; also in the untreated cells, calbindin-D^K level is minimal, (c and d) long-term control (c) and vitamin D-treated (d) cultures, showing distinct mat uration and hypertrophy, with gradual dissolution of the yolk granules, in the endodermal cells as a function of culture time and the substantial stimulation of calbindin-D28K ex pression by vitamin D. Bar = 50 fan.
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cells (Fig. 5d). Calbindin immunostaining was gener ally evenly spread out in the cytoplasm, with the excep tion of a small peripheral zone. When yolk sac endodermal cells were obtained from d 6 embryos, the cultured cells showed significant yolk internalization activity (Fig. 6). In both short-term and long-term cultures, calcitriol treatment stimulated calbindin-D28Kexpres sion (Fig. 6b and d). Finally, cultured yolk sac endoderrnal cells from d 9 embryos showed the highest activity of yolk internalization (Fig.7a and b). Calbindin expres sion was again significantly stimulated by calcitriol treatment in both short-term and long-term cultures (Fig. 7b and d). Taken together, these results clearly demonstrated the cell-specific, stimulatory action of calcitriol on calbindin-D28Kexpression by yolk sac endodermal cells. The calcitriol-dependent stimulation of calbindinDISKexpression in the yolk sac was further examined at the level of protein and mRNA. As shown in the Western immunoblots in Figure 8, analysis of calbindin-D28Klevels in d 3 and d 6 yolk sac specimens pro cessed via various combinations of calcitriol treatment regimens (i.e., varying lengths of treatment in vivo and/ or in vitro) clearly showed that the hormone consis tently up-regulated calbindin-D28Kexpression. It also appeared that 2 d of calcitriol treatment in vivo resulted in maximal calbindin-D2gKexpression, which was not further increased by additional in vitro exposure to calcitriol (e.g., compare lanes 5 and 6 in both Figs. 8A and B). As shown in Figure 9A, the stimulatory effect of calcitriol on calbindin-D28Kexpression was also de tected at the mRNA level for yolk sacs from d 3, d 6, as well d 9 embryos. The response appeared to be high est in the d 9 yolk sac (compare lane 5 to lane 6). To
investigate the molecular basis of the stimulatory ef fect of calcitriol on calbindin-D28Kexpression, the yolk sac RNA blot was reprobed for vitamin D receptor. The results in Figure 9C showed that the level of vitamin D receptor mRNA actually decreased slightly during development (compare lanes 1, 3 and 5 as normalized using the corresponding 0-actin mRNA levels in Fig. 9B); furthermore, calcitriol treatment did not signifi cantly change the expression of the vitamin D receptor.
DISCUSSION In this report, we have demonstrated that chick em bryonic yolk sac endodermal cells are highly responsive to calcitriol, the active metabolite of vitamin D3. Fur thermore, yolk sac calbindin-D28Kexpression and its regulation by calcitriol appear to be in place as early as d 3 of incubation during chick embryonic develop ment, as detected by immunohistochemistry, Western immunoblotting and Northern mRNA analysis. That the yolk sac endoderm is indeed a target tissue for vita min D is further supported by the expression of the vitamin D receptor; however, the calcitriol effect on calbindin-D28Kdoes not appear to require a concomi tant stimulation of vitamin D receptor expression. In previous studies by us (Ono and Tuan 1991) and others (Clark et al. 1989, Lee and Clark 1993),the yolk sac calcium-mobilizing activity and its regulation by vitamin D has been clearly demonstrated. Extensive investigations of intestinal calcium transport and the regulatory action of vitamin D (see Wasserman and Fullmer 1989) firmly established the functional
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FIGURE 8 Vitamin D stimulation of calbindin-D28Kexpression in chick embryonic yolk sac examined by Western immunoblot. Yolk sac samples were processed for calcitriol treatment in ovo and in vitro as described in Figure 1, and proteins were extracted and prepared for Western immunoblot as described in Materials and Methods. SL = shell-less culture of whole embryos. (A] std., prestained high molecular weight protein standard; lane 1, 3 d in ovo + 3 d organ culture; lane 2, 3 d in ovo + 1 d organ culture + 2 d calcitriol organ culture,- lane 3, 3 d in ovo + 2 d SL -l-1 d organ culture; lane 4, 3 d in ovo + 2 d SL + 1 d calcitriol organ culture,- lane 5, 3 d in ovo + 2 d calcitriol SL + 1 d organ culture; lane 6, 3 d in ovo + 2 d calcitriol SL + 1 d calcitriol organ culture. (B) std., prestained high molecular weight protein standard; lane 1, 6 d in ovo + 3 d organ culture,lane 2, 6 d in ovo + 1 d organ culture + 2 d calcitriol organ culture; lane 3, 6 d in ovo + 2 d SL + 1 d organ culture; lane 4, 6 d in ovo + 2 d SL + 1 d calcitriol organ culture,- lane 5, 6 d in ovo + 2 d calcitriol SL + 1 d organ culture; lane 6, 6 d in ovo + 2 d calcitriol SL + 1 d vitamin D organ culture. Protein loads were 12 ¿ig/lane. Note all samples showed significant stimulation of calbmdin-D28Kexpression as a result of vitamin D treatment. It appears that 2 d of vitamin D treatment in vivo resulted in maximal expression that was not further increased by additional in vitro exposure to vitamin D (compare lanes 5 and 6).
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FIGURE 9 Vitamin D Stimulation of calbindin-D28K and vitamin D receptor expression in chick embryonic yolk sac examined by Northern mRNA hybridization. (A} calbindin; (B) ß-actin; (C) vitamin D receptor Northern blots. SL = shell-less culture of whole embryos, lane 1, 3 d in ovo + 3 d SL culture; lane 2, 3 d in ovo + 3 d SL culture with calcitrici; lane 3, 3 d in ovo + 6 d SL culture,- lane 4, 3 d in ovo + 3 d SL + 3 d calcitriol SL; lane 5, 3 d in ovo + 9 d SL culture,lane 6, 3 d in ovo + 6 d SL + 3 d calcitriol SL. (A ) Note the significant stimulation of calbindin-D2«KmRNA (~2 kb) by vitamin D for all developmental stages. Also note that a higher molecular weight mRNA (~2.6 kb) is also seen in some vitamin D-treated yolk sac samples. (C) Note the changing (decreasing) level of vitamin D receptor as a func tion of development; however, vitamin D treatment did not result in significant changes in the vitamin D receptor level at each of the developmental stages.
involvement of the cytosolic, vitamin D-dependent, Ca2+-binding protein, calbindin-D28K, in cellular cal cium handling. Although the exact role of calbindinD28Kremains incompletely understood, it most likely participates in the facilitated diffusion of calcium in the transporting epithelium (Feher et al. 1992). The presence of calbindin-D28Kin the yolk sac endoderm thus strongly suggests a similar functional role for calbindin-D28Kin the mobilization of yolk calcium during embryonic development. The temporal profiles of calcium content in the vari ous embryonic and extraembryonic compartments of the chicken egg during incubation (Johnston and Comar 1955, Packard and Packard 1984, Simkiss 1961) have been used to formulate the following sequence of
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calcium mobilization by the developing embryo (Tuan 1987, Tuan et al. 1991):Phase 1 (d 7-10), when calcium is mobilized exclusively from the yolk; Phase 2 (d 1014), when the eggshell calcium reserve begins to be mobilized by the chorioallantoic membrane; Phase 3 (d 14-20), when active chorioallantoic membrane cal cium mobilization continues and some of the calcium from the eggshell is temporarily stored in the yolk; and Phase 4 (d 20 to hatching), when the chorioallantoic membrane calcium transport function ceases and the entire yolk is retracted. The vitamin D-regulated ex pression of calbindin-D28Kin the d 9 yolk sac is thus compatible with this general scheme. In fact, in our previous study (Ono and Tuan 1991), we found that calcitriol stimulation of calbindin-D28Kexpression is relatively higher in d 9 yolk sac in comparison with d 14 yolk sac, consistent with the findings by Lee and Clark (1993) that vitamin D stimulation of yolk sac calcium also exhibits the following pattern: d 9 > d 12 > d 15. An interesting finding from this study is that the d 3 yolk sac also expresses a significant level of calbindinD28K,which is also stimulated upon treatment with calcitriol in vitro or in vivo. Functionally, the early yolk sac serves as the site of vasculogenesis and angiogenesis (Flamme 1989) and of serum protein synthesis (Young et al. 1980). During early embryonic develop ment, the yolk sac endodermal cells, together with the splanchnic mesoderm, migrate rapidly to eventually surround the entire egg yolk by incubation d 6 (Ro manoff 1960). The endodermal cells, which line the internal surface of the yolk sac, are responsible for intemalization of the yolk and are thus the principal nu trient-transporting cell type of the embryo. Because of its continuity with the gut endoderm, the yolk sac is essentially an extension of the embryonic intestine, although no yolk passes into the intestine. A number of transport and nutrition-related functions have been described for the yolk sac, including cholesterol esterfication (Shand et al. 1993), immunoglobulin binding and intemalization (Donaldson et al. 1990)and uptake of egg white proteins (Sugimoto et al. 1989). As dis cussed above, calcium mobilization from the egg yolk probably does not initiate until d 7 of incubation and is generally thought to be also mediated by an endocytosis-like mechanism (Komazaki et al. 1993), although recent findings by Lee and Clark (1993) suggest that other cytoskeleton-mediated cellular mechanisms may also be involved. The expression of calbindin-D28Kin the d 3 yolk sac endoderm therefore poses the interest ing question concerning whether the early yolk sac in fact also actively mobilizes yolk calcium. Because re cent analysis of the gene structure of calbindin-D28Khas revealed a well-defined vitamin D response element in its upstream promoter region (Minghetti et al. 1988 and 1989), another issue concerns how calbindin-D28K expression in the early yolk sac is normally regulated. It is noteworthy that our results clearly demonstrate
CALBINDIN IN EMBRYONIC YOLK SAC
ACKNOWLEDGMENT The authors thank David Kreitzer for excellent assis tance in photographic preparation.
LITERATURECITED Bethke, R. M., Record, P. R., Kick, C. H. & Kennaid, D. C. (1936) Effect of different sources of vitamin D on laying bird. I. Egg production, hatchability, and tissue composition. Poult. Sci. 15: 326-335. Branion, H. D. & Smith, J. B. (1932) The influence of vitamin D on hatchability and egg production. Poult. Sci. 11: 261-265. Chomczynski, P. & Sacchi, N. (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform ex traction. Anal. Biochem. 162: 156-159. Clark, N. B., Murphy, M. J. & Lee, S. K. (1989) Ontogeny of vita min D action on the morphology and calcium transport properties of the chick embryonic yolk sac. J. Dev. Physiol. 11: 243-251. Donaldson, J. G., Bogenman, E. & Roth, T. F. (1990) Cultured chick yolk sac epithelium: structure and IgG surface binding. Eur. J. Cell Biol. 53: 246-254. Enomoto, H., Hendy, G. N., Andrews, G. K. & Clemens, T. L. (1992) Regulation of avian calbindin-D28Kgene expression in primary chick
kidney cells: importance of post transcriptional mechanisms and calcium ion concentration. Endocrinology 130: 3467-3474. Feher, J. A., Fullmer, C. S. & Wasserman, R. H. (1992) Role of facilitated diffusion of calcium by calbindin-D28K in intestinal calcium absorption. Am. J. Physiol. 262: C517-C527. Flamme, I. (1989) Is extra embryonic angiogenesis in the chick embryo controlled by the endoderm? A morphology study. Anat. Embryol. 180: 259-272. Hart, L. & DeLuca, H. (1985) Effect of vitamin D3 metabolites on calcium and phosporus metabolism in chick embryos. Am. J. Physiol. 248: E281-E285. Henry, H. & Norman, A. (1976) Vitamin D: two hydroxylated me tabolites are required for normal chicken egg hatchability. Science 201: 835-837. Johnston, P. & Comar, C. (1955) Distribution of calcium from the albumen, yolk and shell to the developing chick embryo. Am. J. Physiol. 183: 365-370. Juurlink, B. & Gibson, M. (1973) Histogenesis of the yolk sac in the chick. Cañad.J. Zool. 51: 509-519. Komazaki, S., Takada, M. & Clark, N. B. (1993) Ultrastructural localization of calcium in the chick yolk membrane endodermal cells as revealed by cytochemistry and X-ray microanalysis. Anat. Embryol. 187: 607-614. Kram, D. & Klein, N. W. (1976) Serum protein synthesis in the early chick embryo. Dev. Biol. 52: 300-309. Lambson, R. (1970) An electron microscopic study of the endoder mal cells of the yolk sac of the chicken during incubation and after hatching. Am. J. Anat. 129: 1-20. Lee, S. K., Clark, N. B. & Brown, S. C. (1990) Action of 1,25dihydroxyvitamin and D3 and parathyroid hormone on 45calcium uptake by the yolk sac membrane of chick embryos. J. Exp. Zool. 256: 297-302. Lee, S. K. & Clark, N. B. (1993) Effects of cytochalasin B on cal cium transport by 1,25 (OH)iD3- or PTH-treated chick embryonic yolk sac in vitro and in vivo. J. Exp. Zool. 266: 11-18. Mangelsdorf, D., Komm, B., McDonnell, D., Pike, J. W. & Haussler, M. (1987) Immunoselection of cDNAs to avian intestinal cal cium binding protein 28K and novel calmodulin-like protein: as sessment of mRNA regulation by the vitamin D hormone. Bio chemistry 26: 8332-8338. Meyer, J., Fullmer, C., Wasserman, R., Komm, B. & Haussler, M. (1992) Dietary restriction of calcium, phosphorus, and vitamin D elicits differential regulation of mRNAs for avian intestinal calbindin-D28K and 1,25-dihydroxy vitamin D3 receptor. J. Bone Miner. Res. 7: 441-448. Minghetti, P., Cancela, L., Fujisaw, Y., Theofan, G. & Norman, A. W. (1988) Molecular structure of chicken vitamin D-induced calbindin D28Kgene reveals eleven exons, six Ca2+-binding domains, and numerous promoter regulatory elements. Mol. Endocrinol. 2: 355-376. Minghetti, P., Gibbs, P. E. M. & Norman, A. W. (1989) Computer analysis of 1,25-dihydroxy vitamin D3-receptor regulated promot ers: identification of a candidate Di-responsive element. Biochem. Biophys. Res. Commun. 162: 869-875. Mobbs, I. & McMillan, D. (1981) Transport across endodermal cells of the chick yolk sac during early stages of development. Am. J. Anat. 160: 285-308. Nakada, M. & DeLuca, H. F. (1985) The appearance of 1,25-dihy droxy vitamin D.,-receptor during chick embryo development. Arch. Biochem. Biophys. 238: 129-134. Narbaitz, R. (1979] Response of shell-less cultured chick embryos to exogenous parathyroid hormone and 1,25-dihydroxy cholecalciferol. Gen. Comp. Endocrinol. 37: 440-442. Narbaitz, R. & Tsang, C. P. W. (1989) Vitamin D deficiency in the chick embryo: The effects of prehatching motility and on the growth and differentiation of bones, muscles and parathyroid glands. Calcif. Tiss. Int. 44: 348-355. Narbaitz, R., Tsang, C. P. W. & Grunder, A. (1987) The effects of
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the presence of the vitamin D receptor in the early, d 3 yolk sac (Fig. 9); thus the yolk sac may be the earliest embryonic tissue expressing the vitamin D receptor, because standard target tissues, such as the kidney and intestine, exhibit receptor activity only after incuba tion d 12 (Nakada and DeLuca 1985). Finally, the issue of availability of calcitriol to the d 3 yolk sac endoderm must also be considered. The egg yolk is one of the richest natural depots of vitamin D and its metabolites (Romanoff 1967). Given this scenario, the active me tabolites of vitamin D would be conveniently made available to the yolk sac endodermal cells once the yolk is internalized and degraded within the cell, thus activating in turn the expression of calbindin-D28K and the transcellular calcium mobilization process. Cultured yolk sac endodermal cells have been used in many studies concerning the activities of early extraembryonic endodermal cells (e.g., Donaldson et al. 1990, Kram and Klein 1976, Young and Klein 1983). In this study, we have shown that the yolk sac endoder mal cells mature in culture in a manner analogous to their development in vivo, forming large, hypertrophie cells that actively internalize and digest yolk droplets. More importantly, the cultured yolk sac endodermal cells represent a convenient, easily obtainable, embry onic epithelial cell type responsive to the action of vita min D. This system thus joins a recently established primary cell system consisting of kidney epithelial cells isolated from adult, vitamin D-deflcient chickens (Enomoto et al. 1992) as highly useful cultured cell systems for the analysis of the cellular and molecular action of vitamin D on calbindin-D28K expression.
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SUPPLEMENT Sunde, M., Turk, C. & DeLuca, H. F. (1978) The essentiality of vitamin D metabolites for embryonic chick development. Science 200: 1067-1069. Taylor, A. & Wasserman, R. H. (1970) Immunofluorescent local ization of vitamin D-dependent calcium-binding protein. J. Histochem. Cytochem. 18: 107-115. Tuan, R. S. (1980) Calcium transport and related functions in the chorioallantoic membrane of cultured shell-less chick embryos. Dev. Biol. 74: 196-204. Tuan, R. S. (1987) Mechanism and regulation of calcium transpon by the chick embryonic chorioallantoic membrane. J. Exp. Zool. (suppl. 1): 1-13. Tuan, R. S. & Ono, T. (1986) Regulation of extra embryonic cal cium mobilization by the developing chick embryo. J. Embryol. Exp. Morphol. 97: 63-74. Tuan,R.S.,Ono,T.,Akins,R.E.&Koide,M. (1991) Experimen tal studies on cultured, shell-less fowl embryos: calcium transport, skeletal development and cardiovascular functions. In: Egg Incubation: Its Effects on Embryonic Development in Birds and Reptiles (Ferguson, M. & Deeming, D. C., eds.), pp. 419-433. Cambridge University Press, Cambridge, United Kingdom. Wasserman, R. H. & Fullmer, C. (1989) On the molecular mecha nism of intestinal calcium transport. Adv. Exp. Med. Biol. 249: 45-65. Young, M. F. & Klein, N. W. (1983) Synthesis of serum proteins by cultures of chick embryo yolk sac endodermal cells. Dev. Biol. 100: 50-58. Young, M. F., Minghetti, P. P. & Klein, N. W. (1980) Yolk sac endoderm: exclusive site of serum protein synthesis in early chick embryo. Dev. Biol. 75: 239-245.
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vitamin D deficiency in the chick embryo. Calcif. Tiss. Int. 40: 109-113. Ono, T. & Tuan, R. S. (1990) Double staining of immunoblot us ing enzyme histochemistry and India ink. Anal. Biochem. 187: 324-327. Ono, T. & Tuan, R. S. (1991) Vitamin D and chick embryonic yolk calcium mobilization: identification and regulation of ex pression of vitamin D-dependent Ca2*-binding protein, cuihindi nD28Kin the yolk sac. Dev. Biol. 144: 167-176. Packard, M. & Packard, G. (1984) Comparative aspects of calcium metabolism in embryonic reptiles and birds. In: Respiration and Metabolism of Embryonic Vertebrates (Seymour, R., éd.), pp. 155179. W. Junk Publishing, Dordrecht, The Netherlands. Romanoff, A. L. (1960) The Avian Embryo: Structural and Func tional Development. Wiley Publishing, New York, NY. Romanoff, A. L. (1967) Biochemistry of the Avian Embryo. A Quantative Analysis of Prenatal Development. Wiley Publishing, New York, NY. Sato, M. & Tuan, R. S. (1992) Effect of systemic calcium defi ciency on TGF-/3 gene expression in chick embryonic calvarÃ-a. Dev. Dynam. 193: 300-313. Sechman, A., Shimada, K., Saito, N., leda, T. & Ono, T. (1994) Tissue-specific expression of calbindin-D28K gene during ontogeny of the chicken. J. Exp. Zool. 269: 450-457. Shand, ). H., West, D. W., McCartney, R. J., Noble, R. C. & Speake, B. K. (1993) The esterifkation of cholesterol in a yolk sac membrane of the chick embryo. Lipids 28: 621-625. Sim kiss, K. (1961) Calcium metabolism in avian reproduction. Biol. Rev. 36:321-367. Sugimoto, Y., Saito, A., Kusakabe, T., Hori, K. & Roga, K. (1989) Flow of egg white ovalbumin into the yolk sac during embryo genesis. Biochem. Biophys. Acta 992: 400-403.
