We developed and characterized antibodies specific for FGF-2 and used them to locate FGFQ during chick embryo development. A series of micrographs demonstrated the progression of FGF-2 staining during development of the different tissues and organs. FGF-2 was present in the ectoderm covering the entire embryo, muscle cells, nervous system, neural crest cells, and mesonephros. FGF-2 was also present in the limb from initiation of budding through differentiation. The limb ectoderm and subjacent mesoderm showed the strongest immunostaining, with lower levels in the center of the bud. However, the distribution of FGF-2 positive cells in the mesoderm was not homogeneous. This heterogeneity was not due to cell cycle specific distribution of FGF-2 protein, as flow cytometric analysis showed that FGF-2-positive cells were distributed throughout the cell cycle. However, the amount of anti-FGF-2 fluorescence varied most during G1, consistent with the possibility that FGF-2 is low after M phase and increases during G1. A bioassay was used to demonstrate FGF-2 levels in the wing ectoderm were approximately 2.7-fold greater than in the mesoderm. We propose that the location of FGF-2 in the embryo is consistent with a role in epithelial-mesenchymal interactions; in the limb bud it may prevent differentiation and permit limb outgrowth and subsequent expression of patterning events.

Recent studies indicate that one of the major functions of the apical ectodermal ridge (AER) of the embryonic chick limb bud is to maintain mesenchymal cells directly subjacent to it (i.e., cells extending 0.4-0.5 mm from the AER) in a labile, undifferentiated condition. Furthermore, when mesenchymal cells are freed from the AER's influence, either artifically or as a result of normal polarized proximal-to-distal limb outgrowth, they are freed to commence cytodifferentiation. In a preliminary attempt to investigate at a molecular level the mechanism by which the AER exerts its "negative" effect on the cytodifferentiation of subridge mesenchymal cells, we have examined the effect of a variety of agents that elevate cyclic AMP levels on the chondrogenic differentiation of the unspecialized subridge mesoderm of the limb bud in an organ culture system. Dibutyryl- and 8-hydroxy-cyclic AMP elicit a dose-dependent increase in the rate and amount of cartilage matrix formation and a corresponding dose-dependent increase in sulfated glycosaminoglycan accumulation by subridge mesoderm explants. The stimulatory effect of suboptimal concentrations of cyclic AMP derivatives is potentiated by the addition of theophylline. The stimulatory effect is limited to cyclic AMP derivatives, since dibutyryl-cyclic GMP and 5'-AMP have no effect. Thus agents that elevate intracellular cyclic AMP levels stimulate the chondrogenic differentiation of the unspecialized subridge mesoderm of the embryonic chick limb bud.

Members of the fibroblast growth factor (FGF) family of growth factors are key regulators of limb skeletal patterning and growth. Abnormal expression of FGFs or mutations in their receptors (fgfrs) result in skeletal disorders. Here we show that changes in the expression of f&s are intrinsic properties of differentiating cartilage. In mesenchymal micromass cultures differentiating into cartilage, as in ovo, fgfr 1 mRNA was found predominantly in undifferentiated, proliferating mesenchyme, f& 2 in precartilage cell aggregates, and fgfi-3 in differentiating cartilage nodules. Thus, our data suggest that switches in the expression of fgfr 1, 2, and 3 mRNAs are associated with phases of cartilage patterning both in vitro and in ovo, and mark distinct stages in the development of the limb skeleton. o 1995 Wiley-Lisa, Inc.

FGF-2 protein is present in the ectoderm and mesoderm of the developing chick limb bud. Its importance has been shown by the ability of ectopically applied FGF-2 to replace the apical ectodermal ridge, allowing complete outgrowth and subsequent pattern formation of the limb bud. The first goal of this study was to determine whether FGF-2 mRNA was present in the same ectodermal and mesodermal regions of the chick embryo as FGF-2 protein. FGF-2 also has an antisense message that is convergently transcribed from the opposite DNA strand (Kimelman and Kirschner 119891 Cell 59687496; Volk et al.[1989] EMBO J. 82983-2988). The second goal was to demonstrate the expression and distribution of the antisense message. Using RNAse protection assays we detected a full length protected fragment that corresponds to chick embryo FGF-2 mRNA, and a partially protected fragment that corresponds to the antisense message. We used in situ hybridization to show that FGF-2 mRNA was present in the ectoderm and subjacent mesoderm of the chick wing bud. FGF-2 mRNA was also present in body ectoderm and undifferentiated mesoderm throughout the embryo, and in muscle cells, dorsal neural tube, and mesonephros. In situ hybridization also revealed evidence for the presence of the natural antisense message in the embryo in most, but not all, of the same regions as the FGF-2 mRNA. FGF-2 mRNA and its antisense message colocalized in undifferentiated limb mesoderm; however, antisense message was not detected in differentiated muscle or cartilage. It is important to note that FGF-2 mRNA was always present in the mesonephros but that the antisense message was never observed in the mesonephros, thereby providing an internal control for non-specific signal. Although little is known about its function, Kimelman and Kirschner ([19891 Cell 5 9 687-696) proposed that the antisense message may increase turnover of FGF-2 mRNA. When we compared the in situ hybridization data of both mRNAs with levels of FGF-2 protein (Savage et al.

No abstract

Recent studies indicate that one of the major functions of the apical ectodermal ridge (AER) of the embryonic chick limb bud is to maintain mesenchymal cells directly subjacent to it (i.e. cells extending 0·4–0·5 mm from the AER), in a labile, undifferentiated condition, and that when mesenchymal cells are freed from the AER's influence either artificially or as a result of normal polarized proximal to distallimb outgrowth, they are freed to commence cyto-differentiation. In a preliminary attempt to investigate at a molecular level the mechanism by which the AER exerts its ‘negative’ effect on the cytodifferentiation of subjacent mesenchymal cells, we haveexamined the effect of a variety of agents that elevate cyclic AMP levels on the morphogenesis and differentiation of the unspecialized subridge mesoderm in an organ culture system. In vitro in the presence of the AER, undifferentiated subridge mesoderm explants undergo remarkably normal morphogenesis characterized primarily by progressive polarized proximal to distal outgrowth and changes in the contour of the developing explant. In the presence of cyclic AMP derivatives, explants fail to undergo the polarized outgrowth and contour changes characteristic of control explants. In fact, in the presence of dibutyryl-cyclic AMP and theophylline, AER-directed morphogenesis essentially ceases during the first day of culture. The cessation of AER-directed morphogenesis inthe presence of cyclic AMP derivatives is accompanied by the histochemically and biochemically detectable precocious chondrogenic differentiation of the subridge mesenchymal cells. In control explants, cartilage differentiation only occurs in those proximal cellsof the explant which gradually become located greater than 0·4–0·5 mm from the AER. In contrast, in the presence of cyclic AMP derivatives, cartilage differentiation by cells within 0·4–0·5 mm of the AER is detectable from the first day of culture, and by the third day cartilage formation has occurred throughout the entire explant. Overall, these results indicate that elevating the cyclic AMP content of the subridge mesenchymal cells enables the cells to overcome negative influences on cytodifferentiation and the positive influences on morphogenesis being imposed upon them by the AER. On the basis of this observation and previous studies, a testable model on the role of cyclic AMP in limb morphogenesis and differentiation is proposed.

Previous studies have demonstrated that collagen substrates stimulate in vitro somite chondrogenesis, and that agents that elevate intracellular cyclic AMP levels in hibit the ability of somites to respond to the inductive influence of collagen. In the present investigation, radiommunoassay was utilized to compare the cyclic AMP content of somite explants cultured on purified Type I collagen substrates with control explants cultured on Millipore filters. During the period of culture, the cyclic AMP content of collagen-treated explants is significantly lower than the cyclic AMP content of control explants. The cyclic AMP content of collagen-treated explants is 66% of control values as early as one hour following the initiation of culture, and the cyclic AMP content of collagen-treated explants remains lower than controls throughout the 3-day cultured period. The greatest difference in the cyclic AMP content of collagen-treated and control explants is observed at the seventeenth hour of culture, at which time the cyclic AMP content of collagen-treated explants is 56% of controls. These results combined with previous studies provides support for the hypothesis that collagen elicits a reduction in the cyclic AMP content of embroyic somites and that this reduction is necessary to trigger chondrogenic differentiation.

The cytokinetic properties, specifically the phase-transit times, TG,, Ts, and TGz+M, of chick wing bud cells were estimated using data obtained from continuous labeling of stage 20 embryos with bromodeoxyuridine (BrdUrd). The presence of BrdUrd was detected with monoclonal antibodies, and the amount of DNA in the cells was determined with propidium iodide. The fraction of cells in each cell cycle phase, the fraction of labeled cells, and the relative movement, a measure of the mean DNA content, of all labeled cells were evaluated using bivariate flow cytometry at successive times following introduction of the label. Equations are presented to describe the fraction of unlabeled cells in G, + M, which gives a direct estimate of TG,+M; the fraction of all labeled cells, which can then be used to estimate TG,;. and, finally, the relative movement, which provides an estimate of Ts. Thus, the data measured in these experiments together provide estimates of the progression through the cell cycle of limb mesoderm cells.