Developmental changes in glycosaminoglycans,collagen and collagenase activity in embryonic chick skin

In order to study remodelling of connective tissue during development, changes in glycosaminoglycans, collagen and collagenase activity in embryonic chick skin at various stages have been studied.Collagen content in the skin increased rapidly during days 14 to 18, then leveled off until hatching. Prior to the increase of collagen deposition in the skin, a sharp decrease in chondroitin sulfate was observed between days 11 and 14, while dermatan sulfate increased almost 4 fold during days 12 to 14, then increased steadily until hatching. Hyaluronic acid decreased progressively during the stages investigated (days 11 to 20).At the same stage as the rate of collagen deposition in the tissue became maximal (day 16), the amount of dialyzable hydroxyproline showed a maximum indicating that an increased rate of collagen deposition in the tissue was accompanied by accelerated collagenolysis.Culture of skin from various stages of embryonic development revealed that 16 day old tissue was potentially capable of secreting the highest levels of collagenase. This collagenase was mostly inactive against soluble collagen and collagen fibrils but could be activated by 3 M NaSCN treatment.     

Developmental changes in glycosaminoglycans, collagen and collagenase activity in embryonic chick skin.

In order to study remodeling of connective tissue during development, changes in glycosaminoglycan, collagen and collagenase activity in embryonic chick skin at various stages have been studied. Collagen content in the skin increased rapidly during days 14 to 18, then leveled off until hatching. Prior to the increase of collagen deposition in the skin, a sharp decrease in chondroitin sulfate was observed between days 11 and 14, while dermatan sulfate increased almost 4 fold during days 12 to 14, then increased steadily until hatching. Hyaluronic acid decreased progressively during the stages investigated (days 11 to 20). At the same stage as the rate of collagen deposition in the tissue became maximal (day 16), the amount of dialyzable hydroxyproline showed a maximum, indicating that an increased rate of collagen deposition in the tissue was accompanied by accelerated collagenolysis. Culture of skin from various stages of embryonic development revealed that 16 day old tissue was potentially capable of secreting the highest levels of collagenase. This collagenase was mostly inactive against soluble collagen and collagen fibrils but could be activated by 3 M NaSCN treatment.     

Cell-surface glycosyltransferases in gastrulating chick embryos. I. Temporally and spatially specific patterns of four endogenous glycosyltransferase activities.

Seven classes of endogenous cell-surface glycosyltransferase activities have been investigated in gastrulating chick embryos. Galactosyl-, N-acetylglucosaminyl-, fucosyl-, and sialyl-transferases show temporally and spatially specific autoradiographic patterns that are characteristic for each transferase class. Invaginating primitive streak cells, primordial germ cells, and cranial neural crest cells demonstrate some of the most intense extracellular glycosyltransferase activity. Glucosyl-, N-acetylgalactosaminyl-, and glucuronyltransferases are relatively inactive under these assay conditions. A model is proposed that suggests that embryonic cells migrate over carbohydrate substrates via surface transferase binding to exposed oligosac-charide side chains. One prediction of this model is that sugar nucleotides may be teratogenic. Preliminary experiments support such a prediction. Embryos incubated with UDPgal and UDPglcNAc develop abnormally in ways that are consistent with the locations of some transferase classes. The free sugars, galactose, and N-acetylglucosamine, as well as one of the inactive sugar nucleotides, are relatively ineffective in disturbing normal morphogenesis.     

Developmental changes in the ability to express embryonic pepsinogen in the stomach epithelia of chick embryos

Summary The avian stomach is subdivided into two parts, the proventriculus and the gizzard. It has been shown that the gizzard epithelium can express embryonic chick pepsinogen (ECPg) antigen, a marker protein of the proventricular epithelium, as well as normal proventricular epithelium, under the appropriate experimental conditions. To study the possible mechanisms involved in the suppression of ECPg synthesis in the gizzard epithelium during normal development, we carried out heterotypic and heterochronic recombination experiments of the epithelium and mesenchyme of these two organ rudiments. When recombined and cultured with 6-day proventricular mesenchyme, gizzard epithelium of 3.5- to 12-day embryos expressed pepsinogen at all stages tested. However, the ratio of ECPg-positive cells to total epithelial cells in the gizzard epithelium decreased rapidly when epithelium older than 7 days was cultured with proventricular mesenchyme. In contrast to proventricular mesenchyme, 6-day gizzard mesenchyme did not allow ECPg expression in associated proventricular epithelium of 3.5- to 7-day embryos. These results indicate that gizzard epithelium does not express pepsinogen in normal development because of both a decrease in ability to express the enzyme in itself in the course of development and a repressive influence of gizzard mesenchyme.     

Intestinal epithelial cell differentiation-related changes in glycosyltransferase activities in rats

Intestinal epithelial cells differentiate as they migrate from the crypt-to-villus tip. A ten-fraction crypt-to-villus gradient of epithelial cells from rat small intestine was prepared and homogenates assayed for three glycosyltransferases involved in elongation of asparagine-linked oligosaccharides. The N-acetylglucosaminyltransferases I and II (enzymes which attach N-acetylglucosamine to either the 3' or 6' core mannose, respectively) were assayed with structurally-defined glycopeptides as specific acceptors and galactosyltransferase was assayed with asialo, agalactosylfetuin (galactose is attached to exposed N-acetylglucosamine termini). Inhibitors of glycosidases and pyrophosphatases were included in the assays to minimize effects of breakdown of substrate or product. The results indicate the N-acetylglucosaminyltransferase I shows a gradient of activity increasing from a low at the villus tip to a peak in the lower crypt region. In contrast, N-acetylglucosaminyltransferase II showed two peaks of activity, one in the villus zone and another in the upper crypt region. Galactosyltransferase activity also defined a gradient quite similar to that observed for N-acetylglucosaminyltransferase I, its specific activity being highest in the crypt cells. The specific activity levels of the three enzymes correlated with the apparent order of their action: N-acetylglucosaminyltransferase I much less than N-acetylglucosaminyltransferase II much less than galactosyltransferase. These results suggest a developmental regulation of the glycosyltransferases involved in oligosaccharide chain elongation of glycoproteins during intestinal cell differentiation.     

Developmental changes in carboxylase activities in in vitro cultured coconut zygotic embryos: comparison with corresponding activities in seedlings

Phosphoenolpyruvate Carboxylase (PEPC; EC: 4.1.1.31) and Ribulose 1,5-bisphosphate Carboxylase/Oxygenase (RubisCO; EC: 4.1.1.39) enzyme specific activities were measured during the in vitro development of coconut (Cocos nucifera L.) zygotic mature embryos into plantlets and compared with those of palms produced by conventional seed germination. At the time of initiation of germination, high PEPC and low RubisCO activities were measured in both cultured and conventionally germinated embryos, thus indicating an anaplerotic CO₂ fixation. During both in vitro and in planta development, RubisCO progressively took over and became the main route for inorganic carbon fixation. The in vitro-grown coconut plantlets showed a faster decrease in their PEPC:RubisCO ratio than the seedlings, suggesting that an earlier transition from a heterotrophic to an autotrophic mode of carbon fixation takes place in the in vitro-derived material. Just before acclimatization, the RubisCO activity in in vitro-derived plantlets (2.83 μmol CO₂h⁻¹mg⁻¹ _TSP) was lower than that in seedlings (6.98 μmol CO₂h⁻¹mg⁻¹ _TSP) of the same age. Nevertheless, after acclimatization, RubisCO activities were comparable in both in vitro and in planta germinated material This revised version was published online in June 2006 with corrections to the Cover Date.     

Further characterization of collagen galactosyltransferase from chick embryos.

Optimum extraction of collagen galactosyltransferase activity from chick embryos required relatively high concentrations of detergent and salt. The activity was inhibited by concanavalin A, and the enzyme had a high affinity for columns of this lectin coupled to agarose; these results suggest the presence of carbohydrate units in the enzyme molecule. Collagen galactosyltransferase was highly labile, and only 1% of the originally bound enzyme activity could be eluted from the concanavalin A-agarose column with a buffer containing methyl glucoside and ethylene glycol. The purification of the activity over the original supernatant of chick embryo homogenate was 250-300-fold, with the optimum reaction conditions for the purified transferase differing somewhat from those for crude enzyme preparations. The reaction was inhibited by glucose-free basement-membrane collagen, UDP and galactosylhydroxylsine, and also by Co2+ and a number of compounds resembling UDP-galactose. Hydroxylysine was also a weak inhibitor. Immobilized hydroxylysine and UDP-glucuronic acid did not bind the collagen galactosyltransferase, but the enzyme was retarded in a column of UDP-galacturonic acid linked to agarose.     

Developmental acquisition of type X collagen in the embryonic chick tibiotarsus

The temporal and spatial distribution of short chain skeletal (Type X) collagen was immunohistochemically examined in the chick tibiotarsus from 6 days of embryonic development to 1 day posthatching. The monoclonal antibody employed (AC9) was recently produced and characterized as being specific for an epitope located within the helical domain of the type X collagen molecule (T. M. Schmid and T. F. Linsenmayer, J. Cell Biol., in press). The earliest detectable appearance of type X collagen was at 7.5 days, at which time it was restricted to a middiaphyseal location (i.e., in the primary center of ossification). This was in marked contrast to type II collagen, which appears earlier and is distributed throughout the cartilaginous anlagen. With increasing embryonic age, the reactivity with the type X antibody progressively extended toward the epiphyses, lagging somewhat behind the progression of chondrocyte hypertrophy. The anti-type X collagen antibody also reacted with the bony matrix itself, but the immunofluorescent signal produced by this source was considerably less than that produced by cartilage. At 19 days of development, a new small site of type X deposition was initiated in an epiphyseal location, which subsequently enlarged in circumference. These results are consistent with our previous biochemical studies suggesting that, in cartilage, type X collagen is specifically a product of that population of chondrocytes which have undergone hypertrophy.     

Partial purification and characterization of collagen galactosyltransferase from chick embryos.

Collagen galactosyltransferase was purified 50-150-fold from chick-embryo extract. The tissue homogenate was prepared in the presence of Triton X-100, since the addition of the detergent doubled the enzyme activity in the homogenate and the extract. Three species of the enzyme activity with different molecular weights were recovered on gel filtration, the mol.wts. being about 450000, 200000 and 50000. Collagen galactosyltransferase activity was strongly inhibited by p-mercuribenzoate, and stimulated by the addition of dithiothreitol to the incubation system. Studies on substrate requirements indicated that denatured citrate-soluble collagen is a more effective substrate than gelatinized insoluble collagen, as judged from their Km values. Experiments on three peptide fractions prepared from citrate-soluble collagen indicated that a fraction with an average mol.wt. of 500-600 contained peptides large enough to meet a minimun requirement for interaction with the enzyme. However, longer peptides were clearly better substrates. When native and heat-denatured citrate-soluble collagens were compared as substrates, practically no synthesis of galactosylhydroxylysine was found with native collagen. This finding suggests that the triple-helical conformation of collagen prevents the galactosylation of hydroxylysine residues.     

Isolation of collagen glucosyltransferase as a homogeneous protein from chick embryos

Collagen glucosyltransferase was isolated as a homogeneous protein from chick embryos by a procedure consisting of ammonium sulphate fractionation, two affinity chromatographies and two gel filtrations. The specific activity of the purified enzyme was 32,000 times that of the 15,000 x g supernatant of the embryo homogenate, and the enzyme was pure when examined by sodium dodecyl sulphate polyacrylamide gel electrophoresis using three different gel compositions. The molecular weight of the enzyme was about 72,000-78,000 by sodium dodecyl sulphate polyacrylamide gel electrophoresis, the value being dependent on the gel composition. The apparent molecular weight by gel filtration was dependent on the purity and protein concentration. The sedimentation coefficient S20,w was 4.7. The data suggest that the enzyme molecule consists of one polypeptide chain.     

Developmental changes in internal structure of chick heart plasma membrane

Although changes in electrophysiologically measurable membrane properties of chick embryo cardiac plasma membrane have been repeatedly documented during embryonic development, ultrastructural techniques were heretofore too insensitive to detect developmental changes in internal structure of this membrane. We report here significant structural changes detected by applying the quantitative analysis of Kordylewski, Karrison, and Page (Amer. J. Physiol. 245, H992-H997, 1983 and 248, H297-H304, 1985) to stereo imaged electron microscopic negatives of glutaraldehyde-fixed chick embryo hearts, freeze fractured and photographed with a goniometer stage. Between Hamburger-Hamilton stages 12+ (about 48 hr incubation) and 24 (about 96 hr incubation), plasmalemmal P-face particle density of ventricular myocytes increased from 2228 +/- 139 to 3063 +/- 109 (P less than 0.01); thereafter, measurements at stages 30, 37, 40, and 45 (7, 11, 15, and 19 days incubation) showed a slower significant linear increase which gave a least-squares line with a slope of 41 +/- 13 particles/day (P less than 0.01). Just before hatching, (stage 45) the value of 3762 +/- 234 was similar to, though slightly smaller than, the values of 4122 +/- 153 (8 days after hatching) and 4281 +/- 218 (adult chicken). These results indicate striking stage-dependent changes in the population of integral membrane proteins (channels, carriers, receptors, etc.), especially marked during early embryogenesis.     

Developmental changes in the protein kinase activity of chick brain

The protein kinase activity in the postmitochondrial and postribosomal fractions from chick brain at various stages of development was examined. It has been found, that the overall level of protein kinases activity, assayable under the experimental conditions, increases during embryogenesis, sharply decreases at the hatch, and again increases thereafter. The subcellular distribution of protein kinases alters during ontogenetic development. The embryonal protein kinases of both subcellular fractions differ in the protein substrate specificity, cAMP- and salts-sensitivity from those of the adult ones. Thanks to use of ribosomal proteins as an exogenous substrates it was possible to visualize the developmental changes in the protein kinase pattern.     

Quantification of collagen structural changes during chick corneal development

As the most abundant structural mammalian protein, collagen has been implicated in the pathogenesis of numerous diseases such as osteogenesis imperfecta, and cancer. In the case of cornea, abnormal cornea development can lead to conditions such as agenesis, megalocornea, microcornea, and cornea plana. Therefore, understanding the mechanisms of collagen assembly during development may contribute to the prevention or treatment of corneal diseases. In this study, we applied fast Fourier transform second harmonic generation microscopy to quantify parameters of corneal structures during chick development. Our results show that both the rotational pitch and overall rotational angle of corneal stroma modulate between E9 and E19. In addition, we found that corneal structures between left and right corneas are highly correlated during development.      

Developmental changes in the calcium currents in embryonic chick ventricular myocytes

Summary Using the patch-clamp technique, we recorded whole-cell calcium current from isolated cardiac myocytes dissociated from the apical ventricles of 7-day and 14-day chick embryos. In 70% of 14-day cells after 24 hr in culture, two component currents could be separated from totalI _Ca activated from a holding potential (V _h) of –80 mV. L-type current (I _L) was activated by depolarizing steps fromV _h –30 or –40 mV. The difference current (I _T) was obtained by subtractingI _L, fromI _Ca.I _T could also be distinguished pharmacologically fromI _L in these cells.I _T was selectively blocked by 40–160 m Ni²⁺, whereasI _L was suppressed by 1 m D600 or 2 m nifedipine. The Ni²⁺-resistant and D600-resistant currents had activation thresholds and peak voltages that were near those ofI _T andI _L defined by voltage threshold, and resembled those in adult mammalian heart. In 7-day cells,I _T andI _L could be distinguished by voltage threshold in 45% (S cells), while an additional 45% of 7-day cells were nonseparable (NS) by activation voltage threshold. Nonetheless, in mostNS cells,I _Ca was partly blocked by Ni²⁺ and by D600 given separately, and the effects were additive when these agents were given together. Differences among the cells in the ability to separateI _T andI _L by voltage threshold resulted largely from differences in the position of the steady-state inactivation and activation curves along the voltage axis. In all cells at both ages in which the steady-state inactivation relation was determined with a double-pulse protocol, the half-inactivation potential (V _1/2) of the Ni²⁺-resistant currentI _L averaged –18 mV. In contrast,V _1/2 of the Ni²⁺-sensitiveI _T was –60 mV in 14-day cells, –52 mV in 7-dayS cells, and –43 mV in 7-day NS cells. The half-activation potential was near –2 mV forI _L at both ages, but that ofI _T was –38 mV in 14-day and –29 mV in 7-day cells. Maximal current density was highly variable from cell to cell, but showed no systematic differences between 7-day and 14-day cells. These results indicate that the main developmental change that occurs in the components ofI _Ca is a negative shift with, embryonic age in the activation and inactivation relationships ofI _T along the voltage axis.