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Notes: FSR (fragmented sarcoplasmic reticulum) isolated from chicken breast muscle (Pectoralis major) at 0 hr, 48 hr and 7 day postmortem was purified using linear density gradient centrifugation. The Ca++accumulating ability of the FSR was found to increase with postmortem aging. No loss in ATPase activity was noted nor was any significant change observed in the SDS-gel electrophoresis patterns of the proteins with postmortem aging. The FSR from the aged muscle contained a higher proportion of phospholipids These studies indicate that the Ca++ sequestering properties of sarcoplasmic reticulum from chicken breast muscle are not impaired during postmortem aging.

Notes: Summary The mechanism of electrical coupling between cells of earlyXenopus embryos has been studied by examination of the nonjunctional membrane resistances and capacitances as a function of cleavage stage, the junctional and nonjunctional membrane resistances as functions of time during the first cleavage, and the electrical properties of the primitive blastocoel. The changes in membrane resitances and capacitances during the first two cleavages may be completely explained by the addition of new membrane, identical in specific resistance and capacitance to the original membrane, at a constant rate to furrows which are electrically connected to the perivitelline space. Microelectrode recording from the primitive blastocoel indicates that there is no electrical difference detectable between it and the perivitelline space. These results are discussed in the context of current theories of the mechanism of intercellular electrotonic coupling.

Notes: Summary The endocytosis of cationized ferritin and of a phosvitinferritin conjugate by cells of the chick embryo area pellucida has been examined. Cationized ferritin was bound mainly to the free surface of the epiblast but was absent from the region of the primitive streak. The binding was patchy and experiments suggest that the anionic sites which bind cationized ferritin are themselves naturally clustered. Uptake of cationized ferritin was exclusively by coated pits. The resulting coated vesicles delivered the cationized ferritin to membrane-bound sites of accumulation in the cytoplasm and to the close vicinity of Golgi bodies. The cationized ferritin was frequently found to share intracellular vacuoles with yolk granules. The uptake was not affected by the presence of microfilament or microtubule-inhibiting agents. Native ferritin, even at concentrations forty times that of cationized ferritin, was not bound or endocytosed. Coated pits in the epiblast were often associated with overlying extracellular yolk granules. This suggested that the yolk might be inducing the formation of the coated pits. The yolk protein phosvitin was coupled to ferritin and this conjugate was found to be endocytosed by coated pits. This uptake was inhibited in the presence of an excess of free phosvitin but not by albumin, indicating some selectivity for phosvitin over other proteins. The phosvitin conjugate was also found sharing intracellular vacuoles with yolk. We conclude that the cells of the area pellucida, and in particular those of the epiblast, have an active coated vesicle uptake system which may be able to selectively endocytose yolk or yolk protein.

Notes: Summary In the chick embryo the final number of somites is achieved at about stage 22 of Hamburger and Hamilton. By this time the neural tube and notochord have reached the tip of the tail bud but some paraxial mesoderm remains unsegmented. In this study using scanning electron microscopy we show that somitomeres are present in this mesoderm. This indicates that the terminal paraxial mesoderm of the tail bud may have the potential to form supplementary somites, though we do not as yet know what prohibits the completion of segmentation to the tip.

Notes: Summary Lectins have been used extensively to detect changes in carbohydrate moieties on the surface of embryonic cells during early development. Peanut agglutinin (PNA) in particular has been used to investigate changes related to cell differentiation. PNA has also been used to differentiate between the rostral and caudal sclerotome halves which have been shown to be functionally different, with neural crest cells and neurites traversing only the rostral half during their migration. In this study, we have sectioned and stained chick embryos between 3 and 8 days of age with PNA to examine the distribution of PNA binding sites associated with the vertebral column during this period and also to determine the fates of the rostral and caudal sclerotome halves. Ultrastructural localisation of PNA-gold conjugate showed that binding sites for this lectin were present intracellularly and extracellularly both on cell surfaces and in the matrix. At the light microscope level, a clear banding pattern emerged after staining with PNA which consisted of alternating light and dark staining along the entire length of the vertebral axis of the embryo. In the younger embryos, a simple banding pattern emerged where the rostral sclerotome half of each segment stained only lightly while the caudal half stained darkly. This banding pattern was present throughout the 6 day period of development and could be traced continuously but grew more complex as the sclerotome cells migrated to surround the notochord and neural tube and as the dorsal root ganglia developed. The rostral sclerotome half was found to contribute to the caudal part of one vertebral body and its neural arch, while the caudal sclerotome half was found to contribute to the intervertebral disc, the rostral half of the next caudal vertebra, and part of its neural arch.

Notes: Summary In a number of species, the floor plate of the developing neural tube and spinal cord has been ascribed specialized functions associated with the patterning of neuronal differentiation. The differentiation of the floor plate itself is believed to be closely related to the presence of the underlying notochord. Grafting experiments have previously shown that in the chick embryo an implanted segment of notochord is capable of inducing the adjacent host neural plate or neural tube to produce an additional floor plate, although the inductive effect diminishes with increasing age of the host. We have examined the potential of notochord to promote the appearance of floor plate-like structures from neural tube tissue in culture. To facilitate this, it was necessary initially to examine the immunoreactivity of the early neural tube and floor plate in situ and in vitro with a panel of antibodies to identify a suitable marker for floor plate differentiation in vitro. In situ, the differentiation of the floor plate was characterized by a lack of immunoperoxidase staining with antibody to neurofilaments and the monoclonal antibody HNK-1 throughout the period examined. This distinguished the floor plate from other regions of the neural tube, and was in contrast to its conspicuous affinity for antibodies to N-CAM and highly sialylated N-CAM, which also stained several closely adjacent regions of the neural tube over the period examined. We also found that oligodendrocytes occurred both in the floor plate and in the flanking ventral neural tube, and that astrocytes were too poorly represented throughout the neural tube at the stages examined to be useful markers of floor plate differentiation. We therefore concluded that only the anti-neurofilament and the HNK-1 antibodies were potentially useful markers for floor plate differentiation. When these antibodies were tested on cells in culture, neural tube tissue showed the presence of neurofilament and HNK-1-positive neurites, while floor plate cultures showed few of these. These distributions were consistent with those demonstrated in situ. However, cells staining positively for N-CAM, sialylated N-CAM and the glial cell markers were relatively sparse in floor plate cultures, suggesting that these epitopes were not retained or were masked in cultured cells. As a result of these experiments, we selected the absence of neurofilament-positive cells as a marker for floor plate differentiation in culture. Co-culture of pieces of neural tube from 3-day embryos with notochord segments resulted in the suppression of neurofilament-positive neurite outgrowth from the former, and the consequent production of tissue with floor plate-like characteristics. The absence of neurites was most marked on the side of the neural tube tissue that was apposed to the notochord. Co-culture of neural tube with other tissues did not produce this effect. We suggest that the neurite suppression by notochord in vitro is analogous to its activity in situ, and that neural tubes from 3-day embryos are still competent to respond to notochordal tissue.

Notes: Summary We have examined the effects on vertebral development of various combinations of somite removal in two day old chick embryos as shown by vertebral formation after a further seven days of incubation. Each combination produced one of a variety of results ranging from completely normal vertebral formation, through fusion of various vertebral elements, to the absence of complete vertebral halves and the formation of hemivertebrae. Assessment of our operating ability showed that we were removing at least 90% of the somitic material and therefore these results suggest that there is a regulating mechanism available to the embryo, at least with regards to vertebral development. When two consecutive somites were removed, vertebrae frequently developed that were lacking certain elements. This suggests that the somitic cells are already determined with regards to formation of specific vertebral elements. Experiments involving the removal of a bilateral pair of somites (a repetitive unit) also provided evidence of a counting mechanism which ensures that the correct number of total vertebrae are present

Notes: Summary In the chick embryo, the tail bud reaches its maximum length at about stage 22 of Hamburger and Hamilton, after which it starts to regress. By this stage the neural tube and notochord extend right to the tip of the tail, but the somites do not do so, the terminal tail bud mesoderm never becoming segmented. The investigation is concerned with analysing why this mesoderm fails to segment. When tail buds were explanted to the chorio-allantoic membrane, they continued to form somites only until the “correct” number had segmented, i.e., the tail bud formed no more somites when isolated from the embryo than it would have formed if undisturbed. Morphological studies suggest that in the normal embryo massive cell death overtakes the tail bud mesoderm before it can segment. It is suggested therefore that cell death may be a contributory factor in preventing segmentation.

Notes: Abstract We examined the effects of transforming growth factor-β1 (TGFβ1) and a neutralizing monoclonal antibody on two phases of early chick embryo development: gastrulation and chondrogenesis. We carried out experiments in vivo and in vitro on mesoderm cells from the gastrulating embryo at day 1, and on sclerotome cells from day 3 embryos, having previously shown that this factor is present among these cells at these stages of development. Addition of the antibody to cultures of these cells produced a dose-dependent decrease in cell outgrowth and spreading and concomitantly reduced fibronectin deposition. In vivo studies of the effects of TGFβ1 on mesoderm during gastrulation were carried out by grafting beads carrying this agent into gastrulating embryos. We used beads of ion-exchange resin as well as hydrolysed polyacrylamide, and found that the grafts produced an accumulation of mesoderm cells around the implant and, at later stages, the formation of enlarged somites. There was no effect on embryonic axis formation. Studies of bromodeoxyuridine (BrdU) incorporation indicated that the mesoderm accumulation was due, at least in part, to an increase in cell proliferation. However, examination of the effect of TGFβ1 on BrdU incorporation by mesoderm during gastrulation and sclerotome cells in vitro indicated in inhibition of cell proliferation, an inconsistency explained in terms of the variation between the in vivo and in vitro conditions. We conclude that TGFβ1 is both appropriately located, and is able, to influence cell proliferation among the mesodermal cell populations during early development, and that this effect contributes to the overall control of mesodermal morphogenesis. Chondrogenesis was studied in vitro using micromass cocultures of sclerotome cells with notochordon a permeable substratum. Under these conditions, the addition of TGFβ1 caused an increase in the deposition of Alcian blue-stainable material, indicating a stimulation of chondrogenesis. We suggest that this result, coupled with the previous demonstration that TGFβ1 is present among the sclerotome cells in the embryo at this time, supports the contention that this factor exerts a regulatory effect on sclerotome cell differentiation.