Rocky S. Tuan

Chondrogenesis of limb bud mesenchyme in vitro: stimulation by cations.

To analyze the nature of cell-cell interactions in chondrogenesis, two cations that influence these interactions, calcium and poly-L-lysine (PL), were tested for their effects on chondrogenesis in vitro. High density cultures of chick limb bud mesenchyme (Hamilton-Hamburger stages 23/24), were exposed to culture media containing calcium (0.6-3.3 mM) or PL (1-10 micrograms/ml). Both cations stimulated chondrogenesis in a dose-dependent manner, and also promoted cartilage formation in normally non-chondrogenic, low cell density cultures. Chondrogenesis was assayed based on cartilage nodule number, [35S]sulfate incorporation, and expression of type II collagen as detected by immunohistochemistry. The calcium effect was not mimicked by other divalent cations (Cd, Co, Ni, Mg, Mn, and Sr). The effect of PL was dependent on its Mr (greater than or equal to 14K) and charge, and was mimicked by poly-D-lysine but not by lysine or other analogs of PL or lysine (epsilon-amino caproic acid, lysozyme, poly-L-arginine, and spermidine). Calcium and PL probably act by different mechanisms since their effects were additive, and required their presence on different days of culture: calcium acted on Day 1, and PL on Day 2. It is proposed that calcium may play a role in the cell aggregation phase of chondrogenesis whereas PL, or a naturally occurring polypeptide of similar nature, may promote chondrogenesis by crosslinking specific anionic components of the cell surface or extracellular matrix.

Calcium deficiency induces expression of cartilage-like phenotype in chick embryonic calvaria

A detailed histological study of the chick embryonic calvarium was carried out to characterize the effect of calcium deficiency on cell differentiation during embryonic bone formation. Calcium deficiency on cell differentiation during embryonic bone formation. Calcium deficient chick embryos, produced by means of long-term shell-less (SL) culture, developed skeletal anomalies. In addition to reduced mineralization as detected by alizarin staining, significant changes were also observed in the extracellular matrix of the embryonic bones. First, the undermineralized matrix of the calvaria of SL embryos appeared to be more acidic as shown by more intense hematoxylin staining of the trabecular regions compared to controls. Secondly, the presence of sulfated proteoglycans was suggested by specific Alcian blue staining of the calvaria of Day 14 SL embryos. In addition, indirect fluorescence immunohistochemistry confirmed the developmental appearance of type II collagen in calcium-deficient calvaria, and localized it to undermineralized regions of the bone. These observations demonstrate the emergence of a chondrogenic phenotype in a typically osteogenic tissue during, and perhaps in response to, severe systemic calcium deficiency in the developing chick embryo.

Regulation of chondrogenesis by heparan sulfate and structurally related glycosaminoglycans

To test the possible involvement of heparan sulfate proteoglycans in chondrogenesis, we have studied the effect of their major glycosaminoglycan (GAG) component, heparan sulfate, and other structurally related GAGs on chondrogenesis in micromass cultures of chick limb bud mesenchyme (Hamburger-Hamilton stages 23/24). Heparan sulfate and several of its analogs (heparin, dermatan sulfate, and dextran sulfate) were found to significantly stimulate cartilage nodule formation; in addition, heparan sulfate and heparin also promoted nodule growth. Chondroitin sulfate did not stimulate chondrogenesis. The activity of the GAGs was dependent on their dose, molecular size, charge, and chemical structure. Similar effects were also observed in micromass cultures derived from stage 25 embryonic wing-tip subridge mesoderm, an enriched population of chondroprogenitor cells. The time of action of the GAGs during culture suggested their involvement in post-cell aggregation events of chondrogenesis, such as the initial expression of the chondrocyte phenotype or the growth of cartilage nodules.

Expression of collagen type transcripts in chick embryonic bone detected by in situ cDNA-mRNA hybridization☆

The development of the chick embryonic calvarium, an intramembranous bone, is characterized by direct differentiation of cranial ectomesenchymal cells into osteoblasts without the formation of a cartilage anlage. Collagen biosynthesis remains predominantly as type I in the calvaria. However, in severely calcium-deficient chick embryos maintained in shell-less (SL) culture, cartilage-specific type II collagen is synthesized by the calvaria. Immunohistochemistry localized the cells expressing type II collagen to undermineralized regions of the SL bone. In this study, collagen gene expression in bones of normal (N) and calcium-deficient SL chick embryos was examined at Incubation Day 14 by in situ cDNA-mRNA hybridization. A critical step in the procedure, which used biotinylated cDNA probes, was the selection of fixation conditions which maximized RNA retention and maintenance of tissue morphology. Tissues fixed in modified Carnoys fixative (58% ethanol, 30% choloroform, 10% acetic acid, 2% formaldehyde) for 2-4 hr at -20 degrees C sectioned well and retained their cell morphology and cytoplasmic RNA. Other treatments important for the procedure included demineralization in 0.25 M HCl and removal of matrix by hyaluronidase digestion. In situ hybridization with type-specific collagen cDNA probes revealed that type II collagen mRNA was present in cells throughout the SL calvaria. More importantly, cells with type II collagen mRNA were also present in N calvaria which do not synthesize the protein. The overall abundance of type II-positive cells in N calvaria was not significantly different from that in SL calvaria, but their distribution throughout the bones differed. In general, the regional distribution of type II cells was inversely correlated with the extent of matrix mineralization. In the N calvaria, cells containing collagen type II mRNA were absent in the extensively mineralized superior zone, but were found in the temporal zone which showed limited mineralization. On the other hand, in the SL calvaria, which were substantially undermineralized overall, cells with type II mRNA were found throughout the tissue. Interestingly, the overall ratio of type I cells to type II cells was approximately 50% higher in N calvaria. These findings suggest that collagen type mRNA expression in the chick embryonic calvarium is correlated with, and perhaps dependent on, the extent of tissue matrix mineralization.

Cetyltrimethylammonium bromide discontinuous gel electrophoresis: Mr-based separation of proteins with retention of enzymatic activity.

A discontinuous polyacrylamide and agarose gel electrophoresis system is presented here which allows the fine separation of proteins based on molecular weight with the concomitant retention of native enzymatic activity. This system, referred to as the CAT gel, uses the cationic detergent cetyltrimethylammonium bromide (CTAB) and includes a stacking gel based on the zwitterion arginine and the buffer N-tris(hydroxymethyl)-methylglycine. The CAT gel system allows specific enzyme histochemical detection and localization of proteins after gel electrophoresis. We present evidence that the CAT system stacked and separated a broad range of proteins into discrete bands which migrate as a linear function of log Mr. We have also assessed the effect of CTAB solubilization on the activity of several proteins and showed that some proteins separated by CAT electrophoresis maintain high levels of native enzymatic activity and may be detected histochemically in situ.

Effect of experimentally induced calcium deficiency on the developmental expression of collagen types in chick embryonic skeleton

In order to investigate the effect of embryonic calcium deficiency on the cellular differentiation processes in embryonic skeletogenesis, chick embryos were maintained in long-term shell-less cultures in vitro. The absence of the eggshell, which normally provides over 120 mg of calcium to the embryo during the course of development, resulted in severely retarded and anomalous skeletal formation. The pattern of cytodifferentiation in the skeletal elements during development was assessed by examining collagen type synthesis in both endochondral and intramembranous bones of normal and shell-less embryos as a function of developmental age. Skeletal tissues obtained from these embryos at various developmental stages were maintained in short-term organ culture in medium containing [3H]Pro. The metabolically labeled collagen was isolated from these tissues and typed biochemically based on electrophoresis, ion-exchange chromatography, differential salt fractionation, zone precipitation chromatography, and CNBr peptide mapping. The results indicate that, compared to chronologically equivalent normal controls, calcium-deficient skeletal elements from shell-less embryos appeared to fail to mature into complete bony tissues and instead exhibited partial cartilage phenotype with the expression of cartilage-specific type II collagen.

Vitamin D and chick embryonic yolk calcium mobilization: identification and regulation of expression of vitamin D-dependent Ca2(+)-binding protein, calbindin-D28K, in the yolk sac.

The developing chick embryo acquires calcium from two sources. Until about Day 10 of incubation, the yolk is the only source; thereafter, calcium is also mobilized from the eggshell. We have previously shown that during normal chick embryonic development, vitamin D is involved in regulating yolk calcium mobilization, whereas vitamin K is required for eggshell calcium translocation by the chorioallantoic membrane. We have studied here the biochemical action of 1,25-dihydroxy vitamin D3 in the yolk sac by examining the expression and regulation of the cytosolic vitamin D-dependent calcium-binding protein, calbindin-D28K. Two types of embryos are used for this study, normal embryos developing in ovo and embryos maintained in long-term shell-less culture ex ovo, the latter being dependent solely on the yolk as their calcium source. Our findings are (1) calbindin-D28K is expressed in the embryonic yolk sac, detectable at incubation Days 9 and 14; (2) the embryonic yolk sac calbindin-D28K resembles that of the adult duodenum in both molecular weight (Mr 28,000) and isoelectric point, as well as the presence of E-F hand Ca2(+)-binding structural domains; (3) systemic calcium deficiency caused by shell-less culture of chick embryos results in enhanced expression of calbindin-D28K in the yolk sac during late development; (4) yolk sac calbindin-D28K expression is inducible by 1,25-dihydroxy vitamin D3 treatment in vivo and in vitro; and (5) immunohistochemistry revealed that yolk sac calbindin-D28K is localized exclusively to the cytoplasm of the yolk sac endoderm. These findings indicate that the chick embryonic yolk sac is a genuine target tissue of 1,25-dihydroxy vitamin D3.

Calcium-activated ATPase of the human placenta: Identification, characterization, and functional involvement in calcium transport

A specific, membrane-bound, Ca2+-activated and Mg2+-dependent adenosine triphosphatase (ATPase) activity is present in the human term placenta. The enzyme activity is fractionated electrophoretically into two distinct forms which correspond to molecular weights of 120,000 and 145,000. Cytohistochemistry localized the Ca2+-ATPase to the chorionic villi of the placental labyrinth, and specific staining was primarily associated with the syncytio- and cytotrophoblast layers as well as the perivascular cells. The enzyme activity is inhibited by phenothiazin and erythrosin B which also significantly inhibit active calcium in vitro by placental microsomal membrane vesicles.

Polyionic regulation of cartilage development : promotion of chondrogenesis in vitro by polylysine is associated with altered glycosaminoglycan biosynthesis and distribution

The development of cartilage nodules in cultures of chick limb bud mesenchyme (Hamburger-Hamilton stages 23/24) is significantly promoted when the culture medium is supplemented with (poly-L-lysine (PL) (M(r) greater than or equal to 14K) (San Antonio and Tuan, 1986. Dev. Biol. 115: 313). Here we present findings consistent with the hypothesis that PL may promote chondrogenesis by interacting electrostatically with sulfated glycosaminoglycans (GAGs): (1) poly-L-ornithine, poly-L-histidine, poly-D,L-lysine, and lysine-containing heteropolypeptides stimulate chondrogenesis in proportion to their contents of cationic residues; (2) the effects of PL are diminished when limb mesenchyme cultures are supplemented with exogenous GAGs, including heparin, dermatan sulfate, and chondroitin sulfate; (3) in high density cultures of limb bud mesenchyme, the release of sulfated macromolecules, but not of proteins in general, into the culture medium was significantly inhibited by PL (398K M(r)) treatment, and a net increase in total GAG content of the PL-treated cultures was observed; and (4) in monolayer cultures of cells derived from other chick embryonic tissues, including liver, skeletal muscle, and calvaria, PL treatment promoted the cell layer-associated retention of sulfated GAG. These effects were not observed using the nonstimulatory, low M(r) PL (4K). Based on the above findings and those from previous studies, it is proposed that PL may promote chondrogenesis by interacting electrostatically with cartilage GAGs, thus trapping the extracellular matrix around the newly emerging cartilage nodules and thereby stabilizing their growth and differentiation.

Alkaline phosphatase conjugated Protein A as a sensitive reagent to immunoscreen an expression cDNA plasmid library: Isolation of cDNA to the calcium-binding protein of the chick embryonic chorioallantoic membrane

A highly efficient immunoscreening procedure has been developed to isolate cDNA clones to the calcium-binding protein (CaBP) of the chick embryonic chorioallantoic membrane (CAM). A library of total CAM cDNA was constructed using the expression plasmid vector, pUC 19. Bacterial clones containing plasmids with CaBP cDNA inserts were detected immunohistochemically based on their expression of hybrid CaBP protein sequences. For immunodetection, nitrocellulose bacterial colony replicas were treated with specific antibodies to the CaBP followed by incubation with Staphylococcus aureus Protein A conjugated with alkaline phosphatase (AP) which served as a secondary immunoreagent. Positive clones were then histochemically identified based on AP enzyme activity. The identity of the immunopositive clones was further verified by in vitro translation of mRNA selected by hybridization to the cloned cDNA. The AP-based immunoscreening procedure yields stable reaction products with relatively low background, and should find general application for isolating specific cDNA clones from expression cDNA libraries.