The effect of growth rate on differentiation of chick embryo limb bud mesenchyme in organ culture

Small explants of limb bud mesenchyme of day chick embryos which form muscle in organ culture synthesize proportionally less protein than DNA than do large explants which form cartilage. Chondrogenesis occurred in the central area of greatest population density in reaggregating limb bud cells, myotubes in areas of lesser density and fibroblasts in the sparsely populated periphery. Small explants grown in microdrops in plastic dishes undergo less cell division and form cartilage, but not muscle. Small explants on lens paper undergo more cell division and form muscle, but not cartilage.     

Effects of molecular oxygen on chick limb bud chondrogenesis

Chondrogenesis is an important process in the development of the embryonic chick limb. If limb buds are dispersed just prior to the initiation of chondrogenic differentiation and their cells seeded densely in culture as three-dimensional "micromasses," some of the cells differentiate to form chondrogenic nodules. These nodules characteristically produce sulfated proteoglycans and type II collagen. Two conditions within the early avian limb core have been linked causatively to the initiation of chondrogenesis: a limitation in the availability of molecular oxygen and a low NAD content of the tissue. The O2 limitation is thought to be responsible for the low NAD level. We examined the effects of molecular oxygen on the NAD content of chick limb-bud cells in micromass culture, the formation of chondrocytic nodules, and the production of type II collagen and sulfated proteoglycans. The NAD content of the cells in the micromasses and the production of type II collagen did not vary greatly as a function of oxygen availability. The development of the nodules was modified, but not eliminated, by high oxygen partial pressure (0.95). It was eliminated by anoxia. Proteoglycan synthesis was decreased significantly by high oxygen tension and its sulfation was also decreased, more so in the wing-bud than the leg-bud cells. The results suggest that in culture, high oxygen tension is compatible with some, but not all, aspects of chondrogenic differentiation of cells from embryonic chick limbs.     

Effects of extracellular matrix components on the differentiation of chick embryo tail bud mesenchyme in culture

The mesenchymal cells of the chick tail bud comprise the remains of Hensen's node and the primitive streak after gastrulation. This mass of cells, situated at the caudal limit of the chick embryo, is morphologically homogeneous but pluripotent, with the ability to differentiate into a variety of tissues that are both ectoderm- and mesoderm-derived elsewhere in the embryo. These tissues include neuroectoderm, neurons, myoblasts and chondrocytes. As the factors regulating the differentiation of tail bud mesenchyme into so many cell types are unclear, and because the extracellular matrix (ECM) is known to have a profound effect on cellular differentiation in many embryonic systems, we studied the differentiation of tail bud mesenchyme explanted onto a variety of different ECM components as substrata. We report that the histogenetic potential of isolated tail buds in culture compares favourably with that in situ. Using various antibody markers, we have demonstrated that tail bud mesenchyme cultured upon different ECM components as substrata is able to differentiate into neurons, neuroepithelium, melanocytes, muscle and cartilage. Laminin and laminin-containing substrata (Matrigel) were found to promote the differentiation of neural crest derivatives (neurons and melanocytes) and neuroepithelial cells; type I collagen promoted both myogenesis and chondrogenesis; while type IV collagen promoted myogenesis only. We have therefore demonstrated that differentiation of tail bud mesenchyme in vitro is substratum-dependent.     

Fibroblast growth factor levels in the whole embryo and limb bud during chick development

A growth factor with properties very similar to fibroblast growth factor (FGF) was detected in the yolk and white of unfertilized chick eggs, and in the limb bud and bodies of Day 2.5 (stage 18)-13 chick embryos using two complementary and highly sensitive biological assays-competition of 125I-a-FGF binding to the FGF receptors of 3T3 cells and stimulation of DNA synthesis in MM14 cells, a permanent mouse skeletal muscle cell line that is dependent upon FGF for proliferation. Further evidence of the similarity of this growth factor to FGF is provided by the finding that biological activity is lost when the material is bound to a heparin-Sepharose column and restored upon elution with 2.5 M NaCl; the 2.5 M NaCl fraction from Day 12 embryos contains several polypeptides of apparent molecular weights 12,500-17,500. The level of FGF in the embryonic chick body is fairly constant between Days 2.5 and 6 (stages 18-29), ranging between 1 and 2 ng FGF/mg protein; but thereafter the level increases so that by Day 13 the body contains about 15 ng FGF/mg protein. In contrast, the level of FGF in the limb but is higher than that in the rest of the body until Day 5 (stage 27); it then undergoes a transient decrease between Days 6 and 7, after which it increases but remains below the level observed in the remainder of the body.     

Pattern regulation in the embryonic chick limb: Supernumerary limb formation with anterior (non-ZPA) limb bud tissue

The formation of supernumerary limbs and limb structures was studied by juxtaposing normally nonadjacent embryonic chick limb bud tissue. A “wedge” (ectoderm and mesoderm) of anterior or mid donor right wing bud (stage 21) was inserted in a slit made in a host right limb bud (stage 21) at the same position as its position of origin or to a more posterior position. The AER of the donor tissue and host wing bud were aligned with each other. Donor tissue was grafted with its dorsalventral polarity the same as the host's limb bud or reversed to that of the host's. Depending on the position of origin of the donor limb bud tissue and the position to which it was transplanted in a host, supernumerary wings or wing structures formed. Furthermore, depending on the orientation of the graft in the host, supernumerary limbs with either left or right asymmetry developed. The results of experiments performed here are considered in light of two current models which have been used to describe supernumerary limb formation: one based on local, short-range, cell-cell interactions and the other based on long-range positional signaling via a diffusible morphogen.     

Appearance of myosin in the chick limb bud

Quantitative microcomplement fixation has been used to detect the appearance of myosin in the chick embryonic limb bud. It has been shown that myosin or a myosinlike molecule is present by stage 23, before muscle can be distinguished histologically.     

Muscle and cartilage differentiation in small and large explants from the chick embryo limb bud

Larger fragments of prospective chondrogenic or myogenic limb bud mesenchyme of the 4-day chick embryo differentiated primarily into cartilage in organ culture. Muscle sometimes was present in the peripheral areas adjacent to the larger central masses of cartilage. When the individual fragments of limb bud mesenchyme were cut into four smaller pieces and grown in organ culture, cartilage did not differentiate but muscle was present.Autoradiographic experiments with labeled thymidine and quantitative experiments with ³H-adenosine revealed a marked stimulation of DNA and RNA synthesis in the smaller explants, compared to the larger masses of limb bud mesenchyme.     

Effects of prolonged antitubulin culture on annulate lamellae in mouse alpha L929 fibroblasts

The fine structure of alpha L929 fibroblasts cultured in colchicine or vinblastine sulfate for periods as long as 48 hr was compared to control cells not exposed to antitubulins . In response to prolonged antitubulin culture, several changes in cell ultrastructure were noted: Control fibroblasts contain cytoplasmic annulate lamellae (AL), but prolonged exposure to either vinblastine sulfate or colchicine results in enhanced development of AL. Single pore complexes are present in the rough-surfaced endoplasmic reticulum (rER) in both control and antitubulin-treated cells, but stacked porous cytomembranes also occur under both conditions. Polyribosomes often are closely associated or continuous with the pore complexes. Many antitubulin-treated cells become multinucleate. Some nuclei in both control and antitubulin-treated cells contain large and multiple nucleoli. The large and multiple nucleoli are either attached directly to the inner membrane of the nuclear envelope or to infoldings of the nuclear envelope. Antitubulin-treated cells, after 48-hr exposure, appear also to contain enhanced quantities of smooth-surfaced endoplasmic reticulum (sER) and cytoplasmic filaments (and in some cells, lysosomes and rER as well) when compared to untreated cells. In both control and colchicine-treated cells, AL can exhibit continuity with either rER or sER. Further, all three membrane systems may at times be continuous, but the quantity of these membranes appears to be greater in colchicine-treated cells than in control cells. The results are discussed with respect to possible functional significance.     

Chick endogenous lectin enhances chondrogenesis of cultured chick limb bud cells

We have studied the effect of β-d-galactoside-specific lectin purified from 14-day-old chick embryos on the differentiation of the mesenchymal cells dissociated from the limb buds of stage 24 chick embryos, using the micro-mass culture method described previously. When the cells were incubated with the lectin during the initial 12 hr of culture, cell proliferation became slightly activated. The lectin-treated cells formed a greater number of cartilage nodules and incorporated about twice as much as [³⁵S]sulfate per cell than the control cultures. The results of this study show that the chick endogenous lectin promotes cartilage differentiation in vitro and that endogenous lectin may possibly be involved in chondrogenesis in vivo.