Osteogenesis in cultures of limb mesenchymal cells

The results of previous reports demonstrated that osteoblasts develop in cultures derived from phenotypically unexpressive stage 24 chick limb mesenchymal cells. The observations reported here suggest that initial cell plating densities may provide environmental conditions deterministic to a particular limb phenotype. Quantitative microscopic studies, histochemical localization of calcium phosphate, and electron microscopy indicate that osteoblasts develop in cultures derived from stage 24 limb mesenchymal cells. Additionally, 1–3% of the cells from stage 24 limbs are associated with mineral deposits when plated at initial high densities (5 × 10⁶ cells per 35-mm culture dish), while more than 50% of the cells are associated with cartilage by Day 9. Cultures plated at intermediate seeding densities (between 2.0 and 2.5 × 10⁶ cells per 35-mm culture dish) have minimal cartilage development, and approximately 20% of the cells are associated with mineral by Day 9. Furthermore, cultures prepared from stage 31 limb mesenchymal cells form well-developed bone nodules with both osteoblasts and osteocytes present, but no cartilage. It is clear from these observations and from a consideration of the initiation of osteogenesisin vivo that the initiation of bone development in the limb is not associated with cartilage development. Based on these studies and observations on the effect of nutrient factors on phenotypic expression in culture, an hypothesis is presented relating differential vascularization and nutrient flow to the determination of limb phenotypesin vivo.     

A search for trade-offs among life history traits inDrosophila melanogaster

Summary Are there underlying developmental and physiological properties of organisms that can be used to build a general theory of life history evolution? Much of the theoretical work on the evolution of life histories is based on the premise of negative developmental and genetic correlations among life history traits. If negative correlations do not exist as a general rule then no general theory taking them into account is possible. Negative genetic correlations among life history traits can come about by antagonistic pleiotropy. One cause of antagonistic pleiotropy is cost allocation trade-offs. Since cost allocation trade-offs are due to underlying physiological constraints they are expected to be common to closely related groups. A second form of antagonistic pleiotropy is specialization of genotypes to different niches. This type of antagonistic pleiotropy is expected to be specific to each population. We looked for trade-offs in life history traits of longevity and fecundity inDrosophila melanogaster. We used a half-sib mating design and raised the offspring at two temperatures, 19°C and 25°C. Correlations between longevity and fecundity showed some evidence of antagonistic pleiotropy at high temperature with no evidence of any trade-offs at low temperature. Correlations of early and late fecundity traits did show evidence of cost allocation trade-offs at both temperatures. Antagonistic pleiotropy was also found for cross-environmental correlations of fecundity traits. We conclude that, although life history trade-offs can not be generally assumed, they are frequently found among functionally related traits. Thus, we provide guidelines for the development of general theories of life history evolution.     

A water-soluble fraction from adult bone stimulates the differentiation of cartilage in explants of embryonic muscle

A water-soluble fraction of a 4 M guanidine HCl extract of demineralized adult bovine bone stimulated the differentiation of cartilage in explants of minced skeletal muscle from embryonic chick legs; cartilage was also induced by a semipurified protein preparation. Cartilage could be identified in treated cultures at 1 week with muscle from day-9 embryos, not before 2 weeks with muscle from day-12 embryos, and not before 3 weeks with muscle from day-19 embryos. The ability to respond to this water-soluble fraction by exhibiting cartilage differentiation was dose-dependent, but not confined to any particular muscle region of the day-12 embryonic leg. These observations indicate that bone-derived soluble chondroinductive agents act on cells in minced embryonic muscle preparations. The induction of cartilage is dependent upon the accessibility of the responding cells to the agents, on the concentration of inductive agents, and on the developmental age of the responsive tissue.     

Fasciation induction by the phytopathogen Rhodococcus fascians depends upon a linear plasmid encoding a cytokinin synthase gene.

Rhodococcus fascians is a nocardiform bacteria that induces leafy galls (fasciation) on dicotyledonous and several monocotyledonous plants. The wild-type strain D188 contained a conjugative, 200 kb linear extrachromosomal element, pFiD188. Linear plasmid-cured strains were avirulent and reintroduction of this linear element restored virulence. Pulsed field electrophoresis indicated that the chromosome might also be a linear molecule of 4 megabases. Three loci involved in phytopathogenicity have been identified by insertion mutagenesis of this Fi plasmid. Inactivation of the fas locus resulted in avirulent strains, whereas insertions in the two other loci affected the degree of virulence, yielding attenuated (att) and hypervirulent (hyp) bacteria. One of the genes within the fas locus encoded an isopentenyltranferase (IPT) with low homology to analogous proteins from Gram-negative phytopathogenic bacteria. IPT activity was detected after expression of this protein in Escherichia coli cells. In R.fascians, ipt expression could only be detected in bacteria induced with extracts from fasciated tissue. R.fascians strains without the linear plasmid but containing this fas locus alone could not provoke any phenotype on plants, indicating additional genes from the linear plasmid were also essential for virulence. These studies, the first genetic analysis of the interaction of a Gram-positive bacterium with plants, suggest that a novel mechanism for plant tumour induction has evolved in R.fascians independently from the other branches of the eubacteria.     

Extravascular fluid dynamics of the embryonic chick wing bud

While a number of models of positional information in the chick wing bud have involved the diffusion of morphogens to establish chemical gradients of morphogenetic activity, only recently have there been attempts to characterize the milieu in which such diffusion must take place. We report an analysis of the fluid dynamics of the extravascular (interstitial) spaces of stage 22-25 chick wing buds, into which aqueous aniline blue dye was microinjected as a visible, unreactive tracer. Six sites along the antero-posterior (A-P) and proximo-distal (P-D) axes were chosen for study. Injections of dye into the posterior half of the wing bud exhibited marked directionality of extravascular transport (mean of all posterior sites = 68%), while anterior injections showed little or no directionality (mean of all anterior sites = 13%). All instances of directed dye movement were disto-proximal, the same direction as the blood flow through the marginal veins. In embryos gassed in situ with CO2, which reversibly stopped the heartbeat and vascular flow, directionality was abolished, yet diffusion rates were unaffected. Posterior disto-proximal extravascular dye movement was correlated with the greater diameter, flow velocity, and volume flow rate of the posterior marginal vein, compared to the anterior marginal vein. Radial diffusion rates were measured, and posterior disto-proximal rates were corrected for measured disto-proximal directionality by the use of a simple diffusion-translation model. Three-way analysis of variance showed that directionality-uncorrected disto-proximal rates in posterior sites were not significantly different from anterior radial rates, but that directionality-corrected posterior rates did differ significantly (P less than 0.0001). A significant stage effect (P less than 0.005) and a significant interaction between the A-P axis and stage (P less than 0.05) were also found. We hypothesize that the spatial arrangement and flow patterns of the vasculature physically determine the fluid dynamics of the interstitium. Based on these observations, we also suggest that disto-proximal extravascular fluid movement in the posterior wing bud presents a barrier to the free diffusion of aqueous molecules, including morphogens originating in the "zone of polarizing activity."     

Double-inhibitor and uncoupler-inhibitor titrations. 1. Analysis with a linear model of chemiosmotic energy coupling

The results of double-inhibitor and uncoupler-inhibitor titrations have been simulated and analyzed with a linear model of delocalized protonic coupling using linear nonequilibrium thermodynamics. A detailed analysis of the changes of the intermediate delta muH induced by different combinations of inhibitors of the proton pumps has been performed. It is shown that with linear flow-force relationships the published experimental results of uncoupler-inhibitor titrations are not necessarily inconsistent with, and those of double-inhibitor titrations are inconsistent with, a delocalized chemiosmotic model of energy coupling in the presence of a negligible leak. Also shown and discussed are how the results are affected by a nonnegligible leak and to what extent the shape of the titration curves can be used to discriminate between localized and delocalized mechanisms of energy coupling.     

The relationship of nicotinamide adenine dinucleotide to the chondrogenic differentiation of limb mesenchymal cells

The nicotinamide adenine dinucleotide (NAD) content of mesenchymal cells from the embryonic chick limb has been hypothesized to control the differentiation of these cells by modulation of ADP-ribosylations. To test this hypothesis, [35S]sulfate incorporation into proteoglycans was monitored as an estimate of the chondrogenic expression of cultured limb mesenchymal cells treated with nicotinamide and nicotinic acid to elevate cellular NAD levels or with nicotinamide and benzamide compounds to inhibit ADP-ribosylations. The results of this study indicated that serum component(s) modulate the interactions between these chemical agents and limb mesenchymal cells and, thus, complicate the interpretations of experiments performed in the presence of serum. With a chemically defined medium that promotes limb mesenchymal cell proliferation and differentiation in vitro, it was demonstrated that: (1) no clear correlation exists between cellular NAD content and the chondrogenic expression of cultured limb mesenchymal cells, (2) nicotinamide and benzamide compounds reduce cell proliferation and, at the higher doses tested, considerably reduce chondrogenesis in limb mesenchymal cell cultures, and (3) limb mesenchymal cells exhibit an enhanced susceptibility to benzamide compounds at a time very early in the culture period which temporally coincides with a transient increase in cellular ADP-ribosylation activity and initial chondrogenic differentiation. These results suggest that NAD does not control the differentiation of limb mesenchymal cells and that ADP-ribosylations are an integral, though not controlling, component of limb mesenchyme cytodifferentiation. A model is presented which proposes a role for ADP-ribosylations during the differentiation of limb mesenchymal cells.     

The establishment of vascular-derived microenvironments in the developing chick wing

The results of previous studies on the temporal sequence of limb vascularization suggest that the prospective myogenic and chondrogenic areas of the mesoderm are distinguished by a differential vascularization pattern prior to the overt expression of muscle- and cartilage-specific phenotypes. The experiments presented here are designed to reveal the dynamic aspects of vascular flow in the limb by the observation of how an inert, particular tracer (india ink) is mobilized and dispersed at specific points in the mesoderm. Data are presented as a temporal sequence of fluid flow "maps" which detail both the rate and the direction of vascular flow in the limb. It is proposed that not only does the vasculature compartmentalize the mesoderm into prospective myogenic and chondrogenic zones but also that these broad areas are subcompartmentalized into discrete microenvironments that are spatially distinct with regard to their capacity for transporting the carbon particles. The developmental significance of this observation may be that limb mesodermal cells are granted precise, "positional" information in the form of the specific nutrient and oxygen levels they encounter during critical, or decisional, phases of morphogenesis.     

Extracellular matrix and the maintenance of the differentiated state: proteoglycans synthesized by replated chondrocytes and nonchondrocytes

It has been previously shown that undifferentiated stage 23 to 24 chick limb bud mesenchymal cells can be maintained in culture under conditions which promote chondrogenesis. As the chondrocytes mature in vitro, their proteoglycan synthesis progresses through a specific and reproducible biosynthetic program. By the eighth day of culture, the chondrocytes are making proteoglycans that are similar to proteoglycans isolated from adult animal tissues. Relative to the Day 8 proteoglycans, the proteoglycans synthesized by chick limb bud chondrocytes earlier in culture have a smaller monomer size, longer chondroitin sulfate chains, shorter keratan sulfate chains, a higher ratio of chondroitin-6-sulfate to chondroitin-4-sulfate, and a decreased ability to interact with hyaluronic acid. We have reported a procedure to remove the cells from Day 8 cultures and strip away most, if not all, of the extracellular matrix. In addition, the chondrocytes can be separated from the 40-50% nonchondrocytic cells normally found in Day 8 cultures, and the two cell populations replated separately. This report describes the analysis of the proteoglycans synthesized by replated cells; this analysis demonstrates quantitative and qualitative differences between chondrocyte and nonchondrocyte proteoglycans. The overall rate of proteoglycan synthesis is fourfold higher and the rate of synthesis of high buoyant density proteoglycans 30-fold higher for replated chondrocytes relative to nonchondrocytes. Qualitatively, more newly synthesized nonchondrocyte proteoglycans partition at lower buoyant density on CsCl equilibrium density gradients than do chondrocyte proteoglycans. Nonchondrocyte proteoglycans are of two major classes: One has a monomer size slightly smaller than that of Day 8 chondrocyte proteoglycan, but has much longer glycosaminoglycan chains. The other is considerably smaller than Day 8 chondrocyte proteoglycans, but has glycosaminoglycans of slightly larger size. In contrast, replated chondrocytes synthesize, even as soon as 4.5 hr after replating, proteoglycans that are identical to Day 8 chondrocyte proteoglycan in monomer size, in glycosaminoglycan chain size, in aggregability, and in the ratio of 6-sulfated to 4-sulfated chondroitin. Since denuding mature Day 8 chondrocytes of their extracellular matrix does not cause them to recapitulate their developmentally regulated program for the biosynthesis of proteoglycans, it is concluded that the quality of mature chondrocyte proteoglycan is not altered by the absence of extracellular matrix.     

Nicotinamide adenine dinucleotide levels in cells of developing chick limbs: possible control of muscle and cartilage development

The studies reported here show that NAD⁺ levels are low in chick limbs which have not yet attained the stage of cellular commitment, that these low levels persist during a time period when major chondrogenic commitment and expression occur, that beyond this stage the NAD⁺ levels in chick limbs rise dramatically and continuously, corresponding to the period of major myogenic development, and that developing cultures of stage 24 mesodermal cells seem to mimic these in vivo events in that myogenic cells are observed when NAD⁺ levels are high and chondrogenic cells are observed when NAD⁺ levels are low. These observations are consistent with the hypothesis that pyridine nucleotides may play some role in the control of muscle and cartilage development in embryonic chick limbs.